Endomannosidases in the modification of glycoproteins in eukaryotes

ABSTRACT

The present invention generally relates to methods of modifying the glycosylation structures of recombinant proteins expressed in fungi or other lower eukaryotes, to more closely resemble the glycosylation of proteins from higher mammals, in particular humans. The present invention also relates to novel enzymes and, nucleic acids encoding them and, hosts engineered to express the enzymes, methods for producing modified glycoproteins in hosts and modified glycoproteins so produced.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/695,243, filed Oct. 27, 2003, which is now U.S. Pat. No. 7,332,299,and which is a continuation-in-part of U.S. application Ser. No.10/371,877, filed on Feb. 20, 2003.

FIELD OF THE INVENTION

The present invention generally relates to methods of modifying theglycosylation structures of recombinant proteins expressed in fungi orother lower eukaryotes, to more closely resemble the glycosylation ofproteins from higher mammals, in particular humans. The presentinvention also relates to novel enzymes and, nucleic acids encoding themand, hosts engineered to express the enzymes, methods for producingmodified glycoproteins in hosts and modified glycoproteins so produced.

BACKGROUND OF THE INVENTION

After DNA is transcribed and translated into a protein, furtherpost-translational processing involves the attachment of sugar residues,a process known as glycosylation. Different organisms produce differentglycosylation enzymes (glycosyltransferases and glycosidases) and havedifferent substrates (nucleotide sugars) available, so that theglycosylation patterns as well as composition of the individualoligosaccharides, even of one and the same protein, will be differentdepending on the host system in which the particular protein is beingexpressed. Bacteria typically do not glycosylate proteins and if so onlyin a very unspecific manner (Moens and Vanderleyden, Arch. Microbiol.168(3):169-175 (1997)). Lower eukaryotes such as filamentous fungi andyeast add primarily mannose and mannosylphosphate sugars, whereas insectcells such as Sf9 cells glycosylate proteins in yet another way. See R.K. Bretthauer et al., Biotechnology and Applied Biochemistry 199930:193-200 (1999); W. Martinet, et al., Biotechnology Letters 199820:1171-1177 (1998); S. Weikert, et al., Nature Biotechnology 1999 17:1116-1121 (1999); M. Malissard, et al., Biochem. Biophys. Res. Comm.2000 267:169-173 (2000); D. Jarvis, et al., Curr. Op. Biotech. 19989:528-533 (1998); and Takeuchi, Trends in Glycoscience andGlycotechnology 1997 9:S29-S35 (1997).

N-linked glycosylation plays a major role in the processing of manycellular and secreted proteins. In eukaryotes, the preassembledoligosaccharide Glc3Man9GlcNAc2 is transferred from dolichol onto theacceptor site of the protein by oligosaccharyltransferase in theendoplasmic reticulum (Dempski and Imperiali, Curr. Opin. Chem. Biol. 6:844-850 (2002)). Subsequently, the terminal a-1,2-glucose is removed byglucosidase I facilitating the removal of the remaining twoa-1,3-glucose residues by glucosidase II (Herscovics, Biochim. Biophys.Acta 1473: 96-107 (1999)). The high mannose glycan remaining isprocessed by the ER mannosidase, to Man8GlcNAc2, prior to translocationof the glycoprotein to the Golgi, where the glycan structure is furthermodified. Incorrect processing of the glycan structure in the ER, inturn, can prevent subsequent modification, leading to a disease state.The absence of glucosidase I results in congenital disorder ofglycosylation type (CDG) IIb which is extremely rare, with only onereported human case, and leads to early death (Marquardt and Denecke,Eur. J. Pediatr. 162: 359-379 (2003)). Isolation of the Chinese hamsterovary cell line Lec23, deficient in glucosidase I, demonstrated that thepredominant glycoform present is Glc3Man9GlcNAc2 (Ray et al., J. Biol.Chem. 266: 22818-22825 (1991)). The initial stages of glycosylation inyeast and mammals are identical with the same glycan structures emergingfrom the endoplasmic reticulum. However, when these glycans areprocessed by the Golgi, the resultant structures are drasticallydifferent, thus resulting in yeast glycosylation patterns that differsubstantially from those found in higher eukaryotes, such as humans andother mammals (R. Bretthauer, et al., Biotechnology and AppliedBiochemistry 30:193-200 (1999)). Moreover, the vastly differentglycosylation pattern has, in some cases, been shown to increase theimmunogenicity of these proteins in humans and reduce their half-life(Takeuchi (1997) supra).

The early steps of human glycosylation can be divided into at least twodifferent phases: (i) lipid-linked Glc3Man9GlcNAc2 oligosaccharidesassembled by a sequential set of reactions at the membrane of theendoplasmatic reticulum (ER); and (ii) the transfer of thisoligosaccharide from the lipid anchor dolichyl pyrophosphate on to denovo synthesized protein. The site of the specific transfer is definedby an Asparagine residue in the sequence Asn-Xaa-Ser/Thr, where Xaa canbe any amino acid except Proline (Y. Gavel et al., Protein Engineering3:433-442 (1990)).

Further processing by glucosidases and mannosidases occurs in the ERbefore the nascent glycoprotein is transferred to the early Golgiapparatus, where additional mannose residues are removed by Golgispecific a-1,2-mannosidases. Processing continues as the proteinproceeds through the Golgi. In the medial Golgi, a number of modifyingenzymes, including N-acetylglucosaminyl-transferases (GnT I, GnT II, GnTIII, GnT IV GnT V GnT VI), mannosidase II, and fucosyltransferases, addand remove specific sugar residues. Finally, in the trans-Golgi,galactosyltranferases and sialyltransferases produce a structure that isreleased from the Golgi. The glycans characterized as bi-, tri- andtetra-antennary structures containing galactose, fucose,N-acetylglucosamine and a high degree of terminal sialic acid giveglycoproteins their human characteristics.

When proteins are isolated from humans or animals, a significant numberof them are post-translationally modified, with glycosylation being oneof the most significant modifications. Several studies have shown thatglycosylation plays an important role in determining the (1)immunogenicity, (2) pharmacokinetic properties, (3) trafficking, and (4)efficacy of therapeutic proteins. An estimated 70% of all therapeuticproteins are glycosylated and thus currently rely on a production system(i.e., host) that is able to glycosylate in a manner similar to humans.To date, most glycoproteins are made in a mammalian host system. It isthus not surprising that substantial efforts by the pharmaceuticalindustry have been directed at developing processes to obtainglycoproteins that are as “humanoid” as possible. This may involve thegenetic engineering of such mammalian cells to enhance the degree ofsialylation (i.e., terminal addition of sialic acid) of proteinsexpressed by the cells, which is known to improve pharmacokineticproperties of such proteins. Alternatively, one may improve the degreeof sialylation by in vitro addition of such sugars by using knownglycosyltransferases and their respective nucleotide sugar substrates(e.g. 2,3 sialyltransferase and CMP-Sialic acid).

Further research may reveal the biological and therapeutic significanceof specific glycoforms, thereby rendering the ability to produce suchspecific glycoforms desirable. To date, efforts have concentrated onmaking proteins with fairly well characterized glycosylation patterns,and expressing a cDNA encoding such a protein in one of the followinghigher eukaryotic protein expression systems:

1. Higher eukaryotes such as Chinese hamster ovary cells (CHO), mousefibroblast cells and mouse myeloma cells (R. Werner, et al.,Arzneimittel-Forschung-Drug rResearch 1998 48:870-880 (1998));2. Transgenic animals such as goats, sheep, mice and others (Dente etal., Genes and Development 2:259-266 (1988); Cole et al., J. Cell.Biochem. 265:supplement 18D (1994); P. McGarvey et al., Biotechnology13:1484-1487 (1995); Bardor et al., Trends in Plant Science 4:376-380(1999));3. Plants (Arabidopsis thaliana, tobacco etc.) (Staub et al., NatureBiotechnology 18:333-338 (2000); McGarvey et al., Biotechnology13:1484-1487 (1995); Bardor et al., Trends in Plant Science 4:376-380(1999));4. Insect cells (Spodoptera frugiperda Sf9, Sf21, Trichoplusia ni, etc.in combination with recombinant baculorviruses such as Autographacalifornica multiple nuclear polyhedrosis virus which infectslepidopteran cells (Altmann, et al., Glycoconjugate Journal 16:109-123(1999)).

While most higher eukaryotes carry out glycosylation reactions that aresimilar to those found in humans, recombinant human proteins expressedin the above mentioned host systems invariably differ from their“natural” human counterpart (Raju, et al. Glycobiology 10:477-486(2000)). Extensive development work has thus been directed at findingways to improving the “human character” of proteins made in theseexpression systems. This includes the optimization of fermentationconditions and the genetic modification of protein expression hosts byintroducing genes encoding enzymes involved in the formation of humanlike glycoforms (Werner et al., Arzneimittel-Forschung-Drug Res.48:870-880 (1998); Weikert et al. Nature Biotechnology 17:1116-1121(1999); Andersen et al., Current Opinion in Biotechnology 5:546-549(1994); Yang et al., Biotechnology and Bioengineering 68:370-380(2000)).

What has not been solved, however, are the inherent problems associatedwith all mammalian expression systems. Fermentation processes based onmammalian cell culture (e.g. CHO, Murine, or more recently, human cells)tend to be very slow (fermentation times in excess of one week are notuncommon), often yield low product titers, require expensive nutrientsand cofactors (e.g. bovine fetal serum), are limited by programmed celldeath (apoptosis), and often do not allow for the expression ofparticular therapeutically valuable proteins. More importantly,mammalian cells are susceptible to viruses that have the potential to behuman pathogens and stringent quality controls are required to assureproduct safety. This is of particular concern since as many suchprocesses require the addition of complex and temperature sensitivemedia components that are derived from animals (e.g. bovine calf serum),which may carry agents pathogenic to humans such as bovine spongiformencephalopathy (BSE) prions or viruses.

The production of therapeutic compounds is preferably carried out in awell-controlled sterile environment. An animal farm, no matter howcleanly kept, does not constitute such an environment. Transgenicanimals are currently considered for manufacturing high volumetherapeutic proteins such as: human serum albumin, tissue plasminogenactivator, monoclonal antibodies, hemoglobin, collagen, fibrinogen andothers. While transgenic goats and other transgenic animals (mice,sheep, cows, etc.) can be genetically engineered to produce therapeuticproteins at high concentrations in the milk, recovery is burdensomesince every batch has to undergo rigorous quality control. A transgenicgoat may produce sufficient quantities of a therapeutic protein over thecourse of a year, however, every batch of milk has to be inspected andchecked for contamination by bacteria, fungi, viruses and prions. Thisrequires an extensive quality control and assurance infrastructure toensure product safety and regulatory compliance. In the case of scrapiesand bovine spongiform encephalopathy, testing can take about a year torule out infection. In the interim, trust in a reliable source ofanimals substitutes for an actual proof of absence. Whereas cells grownin a fermenter are derived from one well characterized Master Cell Bank(MCB), transgenic technology relies on different animals and thus isinherently non-uniform. Furthermore, external factors such as differentfood uptake, disease and lack of homogeneity within a herd may affectglycosylation patterns of the final product. It is known in humans, forexample, that different dietary habits impact glycosylation patterns,and it is thus prudent to expect a similar effect in animals. Producingthe same protein in fewer batch fermentations would be (1) morepractical, (2) safer, and (3) cheaper, and thus preferable.

Transgenic plants have emerged as a potential source to obtain proteinsof therapeutic value. However, high level expression of proteins inplants suffers from gene silencing, a mechanism by which highlyexpressed proteins are down regulated in subsequent generations. Inaddition, it is known that plants add xylose and a-1,3 linked fucose, aglycosylation pattern that is usually not found in human glycoproteins,and has shown to lead to immunogenic side effects in higher mammals.Growing transgenic plants in an open field does not constitute awell-controlled production environment. Recovery of proteins from plantsis not a trivial matter and has yet to demonstrate cost competitivenesswith the recovery of secreted proteins in a fermenter.

Most currently produced therapeutic glycoproteins are thereforeexpressed in mammalian cells and much effort has been directed atimproving (i.e.g., humanizing) the glycosylation pattern of theserecombinant proteins. Changes in medium composition as well as theco-expression of genes encoding enzymes involved in human glycosylationhave been successfully employed (see, for example, Weikert et al.,Nature Biotechnology 17:1116-1121 (1999)).

While recombinant proteins similar to their human counterparts can bemade in mammalian expression systems, it is currently not possible tomake proteins with a humanoid glycosylation pattern in lower eukaryotes(e.g., fungi and yeast). Although the core oligosaccharide structuretransferred to the protein in the endoplasmic reticulum is basicallyidentical in mammals and lower eukaryotes, substantial differences havebeen found in the subsequent processing reactions of the Golgi apparatusof fungi and mammals. In fact, even amongst different lower eukaryotes,there exists a great variety of glycosylation structures. This hasprevented the use of lower eukaryotes as hosts for the production ofrecombinant human glycoproteins despite otherwise notable advantagesover mammalian expression systems, such as: (1) generally higher producttiters, (2) shorter fermentation times, (3) having an alternative forproteins that are poorly expressed in mammalian cells, (4) the abilityto grow in a chemically defined protein free medium and thus notrequiring complex animal derived media components, and (5) and theabsence of retroviral infections of such hosts.

Various methylotrophic yeasts such as Pichia pastoris, Pichiamethanolica, and Hansenula polymorpha, have played particularlyimportant roles as eukaryotic expression systems since because they areable to grow to high cell densities and secrete large quantities ofrecombinant protein. However, as noted above, lower eukaryotes such asyeast do not glycosylate proteins like higher mammals. See, for example,U.S. Pat. No. 5,834,251 to Maras et al. (1994). Maras and Contreras haveshown recently that P. pastoris is not inherently able to produce usefulquantities (greater than 5%) of GlcNAcTransferase I acceptingcarbohydrate. (Martinet et al., Biotechnology Letters 20:1171-1177(1998)). Chiba et al. (J. Biol. Chem. 273: 26298-26304 (1998)) haveshown that S. cerevisiae can be engineered to provide structures rangingfrom Man₈GlcNAc₂ to Man₅GlcNAc₂ structures, by eliminating 1,6mannosyltransferase (OCHI), 1,3 mannosyltransferase (MNN1) andmannosylphosphatetransferase (MNN4) and by targeting the catalyticdomain of a-1,2-mannosidase I from Aspergillus saitoi into the ER of S.cerevisiae, by using a ER retrieval/targeting sequence (Chiba 1998,supra). However, this attempt resulted in little or no production of thedesired Man₅GlcNAc₂. The model protein (carboxypeptidase Y) was trimmedto give a mixture consisting of 27% Man₅GlcNAc₂, 22% Man₆GlcNAc₂, 22%Man₇GlcNAc₂, 29% Man₈GlcNAc₂. As only the Man₅GlcNAc₂ glycans aresusceptible to further enzymatic conversion to human glycoforms, thisapproach is very inefficient for the following reasons: In proteinshaving a single N-glycosylation site, at least 73% of all N-glycans willnot be available for modification by GlcNAc transferase I. In a proteinhaving two or three N-glycosylation sites, at least 93% or 98%,respectively, would not be accessible for modification by GlcNActransferase I. Such low efficiencies of conversion are unsatisfactoryfor the production of therapeutic agents; given the large number ofmodifying steps each cloned enzyme needs to function at highest possibleefficiency.

A number of reasons may explain the inefficiency in the production ofglycan formation mentioned above. This may, in part, be due to theinefficient processing of glycans in the ER either by glucosidase I, IIor resident ER mannosidase. A recently evolved class of mannosidaseproteins has been identified in eukaryotes of the chordate phylum(including mammals, birds, reptiles, amphibians and fish) that is alsoinvolved in glucose removal. These glycosidic enzymes have been definedas endomannosidases. The activity of the endomannosidases has beencharacterized in the processing of N-linked oligosaccharides, namely, inremoving a glucose α1,3 mannose dissacharide. The utility in removing ofthe glucose and mannose residues on oligosaccharides in the initialsteps of N-linked oligosaccharide processing is known to be useful forthe production of complex carbohydrates has been well-established.Although endomannosidases were originally detected in the trimming ofGlcMan₉GlcNAc₂ to Man₈GlcNAc₂, they also process other glucosylatedstructures (FIG. 1). Overall, mono-glucosylated glycans are mostefficiently modified although di- and tri-glucosylated glycans may alsobe processed to a lesser extent (Lubas et al., J. Biol. Chem.263(8):3990-8 (1988)). Furthermore, not only is GlcMan₉GlcNAc₂ is thepreferred substrate but other monoglucosylated glycans, such asGlcMan₇GlcNAc₂ and GlcMan₅GlcNAc₂, are trimmed (to Man₆GlcNAc₂ andMan₄GlcNAc₂, respectively) just as efficiently. The occurrence of thisclass of proteins so late in evolution suggests that this is a uniquerequirement to enhance the pronounced trimming of N-linked glycans, asobserved in higher eukaryotes. This suggestion is further strengthenedby the fact that endomannosidase is located in the Golgi and not the ERwhere complete deglucosylation has traditionally been reported to occur.

Previous research has shown that glucose excision occurs primarily inthe ER through sequential action of glucosidase I and II (Moremen etal., Glycobiology 4: 113-125 (1994)). However, more recent researchsuggests the apparent alternate glucosidase II—independentdeglucosylation pathway involving a quality control mechanism in theGolgi apparatus (Zuber et al., Mol. Biol. Cell. December; 11(12):4227-40 (2000)). Studies in glucosidase II—deficient mouse lymphomacells show evidence of the deglucosylation mechanism by theendomannosidase (Moore et al., J. Biol. Chem. 267(12):8443-51 (1992)).Furthermore, a mouse lymphoma cell line, PHAR2.7, has been isolatedwhich has no glucosidase II activity resulting primarily in theproduction of the glycoforms Glc₂Man₉GlcNAc₂ and Glc₂Man₈GlcNAc₂(Reitman et al., J. Biol. Chem. 257: 10357-10363 (1982)). Analysis ofthis latter cell line demonstrated that, despite the absence ofglucosidase II, deglucosylated high mannose structures were present,thus, indicating the existence of an alternative processing pathway forglucosylated structures (Moore and Spiro, J. Biol. Chem. 267: 8443-8451(1992)). The enzyme responsible for this glucosidase-independent pathwayhas been identified as endomannosidase (E.C. 3.2.1.130). Endomannosidasecatalyzes the hydrolysis of mono-, di- and tri-glucosylated high mannoseglycoforms, removing the glucose residue(s) present and thejuxta-positioned mannose (Hiraizumi et al., J. Biol. Chem. 268:9927-9935 (1993); Bause and Burbach, Biol. Chem. 377: 639-646 (1996)).

The endomannosidase does not appear to distinguish between differingmannose structures of a glucosylated glycoform, hydrolyzingGlc₁Man₉₋₅GlcNAc₂ to Man₈₄GlcNAc₂ (Lubas and Spiro, J. Biol. Chem. 263:3990-3998 (1988)). To date, the only endomannosidase to have been clonedis from the rat liver. Rat liver endomannosidase encodes a predictedopen reading frame (ORF) of 451 amino acids with a molecular mass of 52kDa (Spiro et al., J. Biol. Chem. 272: 29356-29363 (1997)). This enzymehas a neutral pH optimum and does not appear to have any specific cationrequirement (Bause and Burbach 1996, supra). Unlike the glucosidaseenzymes, which are localized in the ER, the endomannosidase is primarilylocalized in the Golgi (Zuber et al., Mol. Biol. Cell 11: 4227-4240(2000)), suggesting that it may play a quality control role byprocessing glucosylated glycoforms leaking from the ER.

Given the utility of modifying glucosylated glycans for the productionof human-like glycoproteins, a method for modifying glucosylated glycansby expressing an endomannosidase activity in a host cell would bedesirable.

SUMMARY OF THE INVENTION

Methods have been developed for modifying a glucosylated N-glycan bygenetically engineering strains of non-mammalian eukaryotes which areable to produce recombinant glycoproteins substantially equivalent totheir human counterparts. These cell lines, including yeast, filamentousfungi, insect cells, and plant cells grown in suspension culture, havegenetically modified glycosylation pathways allowing them to carry out asequence of enzymatic reactions which mimic the processing ofglycoproteins in humans. As described herein, strains have beendeveloped to express catalytically active endomannosidase genes toenhance the processing of the N-linked glycan structures with theoverall goal of obtaining a more human-like glycan structure. Inaddition, cloning and expression of a novel human and mouseendomannosidase are also disclosed. The method of the present inventioncan be adapted to engineer cell lines having desired glycosylationstructures useful in the production of therapeutic proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an endomannosidase modifying mono-, di-and tri-glucosylated glycans in the Golgi in comparison to glucoseprocessing of N-glycans in the ER. Highlighted are additional glucoseresidues that can be hydrolyzed.

FIG. 2 is a schematic diagram of an endomannosidase processing theglucosylated structure Glc₃Man₉GlcNAc₂ to Man₅GlcNAc₂ glycans in theGolgi. Highlighted mannose residues represent constituents which, invarious combinations, produce various types of high mannan glycans thatmay be substrates for the endomannosidase.

FIG. 3 shows a BLAST analysis of rat endomannosidase to identifyhomologues. Panel A shows identification of a human sequence (SEQ IDNO:24) showing 88% identity to the C-terminus of rat endomannosidase(SEQ ID NO:23). Panel B (residues 1-23 of SEQ ID NO:24, aligned withresidues 173-195 of SEQ ID NO:25, respectively in order of appearance)shows the N-terminus of isolated sequence from Panel A which was used toisolate the 5′ region of the human endomannosidase in Panel C. Panel Cshows sequence of the potential N-terminus of human endomannosidase (SEQID NO:25).

FIG. 4 shows nucleotide and amino acid sequences of human liverendomannosidase. Nucleotide sequence (upper) (SEQ ID NO:1) andone-letter amino acid sequence (lower) (SEQ ID NO:2) of humanendomannosidase are shown with residue numbers labeled on the left. Thenucleotide region in bold represents the overlapping segments of Genbanksequences gi: 18031878 (underlined) and gi:20547442 (regular text) usedto assemble the putative full-length human liver endomannosidase. Theputative transmembrane domain identified by Kyte and Doolittle analysis(J. Mol. Biol. 157: 105-132 (1982)) (see FIG. 5) is highlighted by anopen box.

FIG. 5 shows the hydropathy plot of the amino acid sequence of the humanendomannosidase, produced according to the method of Kyte and Doolittle((1982) supra), using the web-based software GREASE and a window of 11residues. The filled-in box represents an N-terminal region of highhydrophobicity, suggesting the presence of a putative transmembranedomain. This region is also represented in FIG. 4 by an open box (aminoacid residues 10-26).

FIG. 6 shows nucleotide and amino acid sequences of mouseendomannosidase (Genbank AK030141). Nucleotide sequence (upper) andone-letter amino acid sequence (lower) of mouse endomannosidase areshown with residue numbers labeled on the left. The putativetransmembrane domain identified by Kyte and Doolittle analysis (J. Mol.Biol. 157: 105-132 (1982)) is highlighted by an open box.

FIG. 7 shows the alignment of three endomannosidase open-reading frames.The human (SEQ ID NO:2), mouse (SEQ ID NO:4) and rat (SEQ ID NO:26)endomannosidase ORFs were aligned using the Megalign software of theDNASTAR suite of programs. The algorithm chosen for the analysis was theCLUSTAL V version (Higgins and Sharp Comput. Appl. Biosci. 5, 151-153(1989)). Residues displayed by shading represent amino acids that areidentical between at least two of the ORFs. The amino acid position ofeach ORF is presented to the left of the aligned sequence.

FIG. 8 depicts a Northern blot analysis of RNAs from a variety of humantissues hybridized with a labeled human endomannosidase nucleic acidprobe.

FIG. 9 depicts a Western blot analysis of prepurification on Ni-resin ofsecreted N-terminal tagged endomannosidase, samples from control (GS115)(A), rEndo (YSH89) (B) and hEndo (YSH90) (C) strains. The samples weredetected using anti-FLAG M2 antibody (Stratagene, La Jolla, Calif.).

FIG. 10A shows a MALDI-TOF MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in P. pastoris RDP-25 (och1 alg3).

FIG. 10B shows a MALDI-TOF MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in P. pastoris RDP-25 (och1 alg3)transformed with pSH280 (rat endomannosidaseΔ48/Mnn11(m)) showing, apeak, among others, at 1099 m/z [c] corresponding to the mass ofMan₄GlcNAc₂ and 1424 m/z [a] corresponding to the mass of hexose 6. Thisstrain was designated as YSH97.

FIG. 10C shows a MALDI-TOF MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in P. pastoris YSH97 after in vitrodigestion with α1,2-mannosidase, exhibiting a peak at 938 m/z [b] (Na⁺adduct) corresponding to the mass of Man₃GlcNAc₂.

FIG. 11A shows a MALDI-TOF MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in P. pastoris RDP-25 (och1 alg3).

FIG. 11B shows a MALDI-TOF MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in P. pastoris RDP-25 (och1 alg3)transformed with pSH279 (rat endomannosidaseΔ48/Van1(s)) showing amongothers, a peak at 1116 m/z [c] corresponding to the mass of Man₄GlcNAc₂and 1441 m/z [a] corresponding to the mass of hexose 6. This strain wasdesignated YSH96.

FIG. 11C shows a MALDI-TOF MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in P. pastoris YSH96 after in vitrodigestion with a 1,2-mannosidase, exhibiting a peak at 938 m/z [b] (Na⁺adduct) corresponding to the mass of Man₃GlcNAc₂ and a second peak at1425 m/z [a] showing a decrease in hexose 6.

FIG. 12A shows a MALDI-TOF MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in P. pastoris RDP-25 (och1 alg3).

FIG. 12B shows a MALDI-TOF MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in P. pastoris RDP-25 (och1 alg3)transformed with pSH278 (rat endomannosidaseΔ48/Gls1(s)) showing, apeak, among others, at 1439 m/z (K⁺ adduct) [c] and a peak at 1422 m/z(Na⁺ adduct) corresponding to the mass of hexose 6 [a]. This strain wasdesignated YSH95.

FIG. 12C shows a MALDI-TOF MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in P. pastoris YSH95 after in vitrodigestion with α1,2-mannosidase, exhibiting a peak at 936 m/z [b] (Na⁺adduct) corresponding to the mass of Man₃GlcNAc₂ and a peak at 1423 m/z[a] showing a decrease in hexose 6.

FIG. 13 shows a high performance liquid chromatogram in vitro assay forrat and human endomannosidase activity. Panel A shows the hexose 6standard GlcMan₅GlcNAc₂ in BMMY. Panel B shows glycan substrate producedfrom rat endomannosidase incubated with supernatant from P. pastorisYSH13. Panel C shows glycan substrate produced from humanendomannosidase incubated with supernatant from P. pastoris YSH16. SeeFIG. 14 for structures corresponding to (i) and (ii).

FIG. 14 represents substrate glycan modification by endomannosidase andsubsequent confirmation of product structure by α1,2-mannosidasedigestion and analysis. Structures illustrated are GlcMan₅GlcNAc₂ (i),Man₄GlcNAc₂ (ii) and Man₃GlcNAc₂ (iii). R represents the reducingterminus of the glycan. The substrate GlcMan₅GlcNAc₂ (i) is modified byan endomannosidase converting it to Man₄GlcNAc₂ (ii) (hydrolyzingGlcα1,3Man). Subsequent α1,2-mannosidase digestion results inMan₃GlcNAc₂ (iii).

FIG. 15 shows a pH profile of the activity of human endomannosidase,indicated as % of GlcMan₅GlcNAc₂ substrate converted to Man₄GlcNAc₂ as afunction of pH.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The methods andtechniques of the present invention are generally performed according toconventional methods well known in the art. Generally, nomenclaturesused in connection with, and techniques of biochemistry, enzymology,molecular and cellular biology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are those wellknown and commonly used in the art. The methods and techniques of thepresent invention are generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification unless otherwise indicated. See, e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al.,Current Protocols in Molecular Biology, Greene Publishing Associates(1992, and Supplements to 2002); Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990); Introduction to Glycobiology, Maureen E. Taylor,Kurt Drickamer, Oxford Univ. Press (2003); Worthington Enzyme Manual,Worthington Biochemical Corp. Freehold, N.J.; Handbook of Biochemistry:Section A Proteins Vol I 1976 CRC Press; Handbook of Biochemistry:Section A Proteins Vol II 1976 CRC Press; Essentials of Glycobiology,Cold Spring Harbor Laboratory Press (1999). The nomenclatures used inconnection with, and the laboratory procedures and techniques of,biochemistry and molecular biology described herein are those well knownand commonly used in the art.

All publications, patents and other references mentioned herein areincorporated by reference.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein, the term “N-glycan” refers to an N-linkedoligosaccharide, e.g., one that is attached by anasparagine-N-acetylglucosamine linkage to an asparagine residue of apolypeptide. N-glycans have a common pentasaccharide core of Man₃GlcNAc₂(“Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers toN-acetyl; GlcNAc refers to N-acetylglucosamine). N-glycans differ withrespect to the number of branches (antennae) comprising peripheralsugars (e.g., fucose and sialic acid) that are added to the Man₃GlcNAc₂(“Man3”) core structure. N-glycans are classified according to theirbranched constituents (e.g., high mannose, complex or hybrid). A “highmannose” type N-glycan has five or more mannose residues. A “complex”type N-glycan typically has at least one GlcNAc attached to the 1,3mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of a“trimannose” core. The “trimannose core” is the pentasaccharide corehaving a Man3 structure. Complex N-glycans may also have galactose(“Gal”) residues that are optionally modified with sialic acid orderivatives (“NeuAc”, where “Neu” refers to neuraminic acid and “Ac”refers to acetyl). Complex N-glycans may also have intrachainsubstitutions comprising “bisecting” GlcNAc and core fucose (“Fuc”). A“hybrid” N-glycan has at least one GlcNAc on the terminal of the 1,3mannose arm of the trimannose core and zero or more mannoses on the 1,6mannose arm of the trimannose core.

Abbreviations used herein are of common usage in the art, see, e.g.,abbreviations of sugars, above. Other common abbreviations include“PNGase”, which refers to peptide N-glycosidase F (EC 3.2.2.18); “GlcNAcTr (1-III)”, which refers to one of threeN-acetylglucosaminyltransferase enzymes; “NANA” refers toN-acetylneuraminic acid.

As used herein, the term “secretion pathway” refers to the assembly lineof various glycosylation enzymes to which a lipid-linked oligosaccharideprecursor and an N-glycan substrate are sequentially exposed, followingthe molecular flow of a nascent polypeptide chain from the cytoplasm tothe endoplasmic reticulum (ER) and the compartments of the Golgiapparatus. Enzymes are said to be localized along this pathway. Anenzyme X that acts on a lipid-linked glycan or an N-glycan before enzymeY is said to be or to act “upstream” to enzyme Y; similarly, enzyme Y isor acts “downstream” from enzyme X.

As used herein, the term “antibody” refers to a full antibody(consisting of two heavy chains and two light chains) or a fragmentthereof. Such fragments include, but are not limited to, those producedby digestion with various proteases, those produced by chemical cleavageand/or chemical dissociation, and those produced recombinantly, so longas the fragment remains capable of specific binding to an antigen. Amongthese fragments are Fab, Fab′, F(ab′)2, and single chain Fv (scFv)fragments. Within the scope of the term “antibody” are also antibodiesthat have been modified in sequence, but remain capable of specificbinding to an antigen. Example of modified antibodies are interspecieschimeric and humanized antibodies; antibody fusions; and heteromericantibody complexes, such as diabodies (bispecific antibodies),single-chain diabodies, and intrabodies (see, e.g., Marasco (ed.),Intracellular Antibodies: Research and Disease Applications,Springer-Verlag New York, Inc. (1998) (ISBN: 3540641513), the disclosureof which is incorporated herein by reference in its entirety).

As used herein, the term “mutation” refers to any change in the nucleicacid or amino acid sequence of a gene product, e.g., of aglycosylation-related enzyme.

The term “polynucleotide” or “nucleic acid molecule” refers to apolymeric form of nucleotides of at least 10 bases in length. The termincludes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNAmolecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA orRNA containing non-natural nucleotide analogs, non-nativeinternucleoside bonds, or both. The nucleic acid can be in anytopological conformation. For instance, the nucleic acid can besingle-stranded, double-stranded, triple-stranded, quadruplexed,partially double-stranded, branched, hairpinned, circular, or in apadlocked conformation. The term includes single and double strandedforms of DNA.

Unless otherwise indicated, a “nucleic acid comprising SEQ ID NO:X”refers to a nucleic acid, at least a portion of which has either (i) thesequence of SEQ ID NO:X, or (ii) a sequence complementary to SEQ IDNO:X. The choice between the two is dictated by the context. Forinstance, if the nucleic acid is used as a probe, the choice between thetwo is dictated by the requirement that the probe be complementary tothe desired target.

An “isolated” or “substantially pure” nucleic acid or polynucleotide(e.g., an RNA, DNA or a mixed polymer) is one which is substantiallyseparated from other cellular components that naturally accompany thenative polynucleotide in its natural host cell, e.g., ribosomes,polymerases, and genomic sequences with which it is naturallyassociated. The term embraces a nucleic acid or polynucleotide that (1)has been removed from its naturally occurring environment, (2) is notassociated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (3) is operatively linkedto a polynucleotide which it is not linked to in nature, or (4) does notoccur in nature. The term “isolated” or “substantially pure” also can beused in reference to recombinant or cloned DNA isolates, chemicallysynthesized polynucleotide analogs, or polynucleotide analogs that arebiologically synthesized by heterologous systems.

However, “isolated” does not necessarily require that the nucleic acidor polynucleotide so described has itself been physically removed fromits native environment. For instance, an endogenous nucleic acidsequence in the genome of an organism is deemed “isolated” herein if aheterologous sequence (i.e., a sequence that is not naturally adjacentto this endogenous nucleic acid sequence) is placed adjacent to theendogenous nucleic acid sequence, such that the expression of thisendogenous nucleic acid sequence is altered. By way of example, anon-native promoter sequence can be substituted (e.g., by homologousrecombination) for the native promoter of a gene in the genome of ahuman cell, such that this gene has an altered expression pattern. Thisgene would now become “isolated” because it is separated from at leastsome of the sequences that naturally flank it.

A nucleic acid is also considered “isolated” if it contains anymodifications that do not naturally occur to the corresponding nucleicacid in a genome. For instance, an endogenous coding sequence isconsidered “isolated” if it contains an insertion, deletion or a pointmutation introduced artificially, e.g., by human intervention. An“isolated nucleic acid” also includes a nucleic acid integrated into ahost cell chromosome at a heterologous site, a nucleic acid constructpresent as an episome. Moreover, an “isolated nucleic acid” can besubstantially free of other cellular material, or substantially free ofculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

As used herein, the phrase “degenerate variant” of a reference nucleicacid sequence encompasses nucleic acid sequences that can be translated,according to the standard genetic code, to provide an amino acidsequence identical to that translated from the reference nucleic acidsequence.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over a stretch of at least aboutnine nucleotides, usually at least about 20 nucleotides, more usually atleast about 24 nucleotides, typically at least about 28 nucleotides,more typically at least about 32 nucleotides, and preferably at leastabout 36 or more nucleotides. There are a number of different algorithmsknown in the art which can be used to measure nucleotide sequenceidentity. For instance, polynucleotide sequences can be compared usingFASTA, Gap or Bestfit, which are programs in Wisconsin Package Version10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences (Pearson, 1990, (hereinincorporated by reference). For instance, percent sequence identitybetween nucleic acid sequences can be determined using FASTA with itsdefault parameters (a word size of 6 and the NOPAM factor for thescoring matrix) or using Gap with its default parameters as provided inGCG Version 6.1, herein incorporated by reference.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 50%, more preferably 60%of the nucleotide bases, usually at least about 70%, more usually atleast about 80%, preferably at least about 90%, and more preferably atleast about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or Gap, as discussed above.

Alternatively, substantial homology or similarity exists when a nucleicacid or fragment thereof hybridizes to another nucleic acid, to a strandof another nucleic acid, or to the complementary strand thereof, understringent hybridization conditions. “Stringent hybridization conditions”and “stringent wash conditions” in the context of nucleic acidhybridization experiments depend upon a number of different physicalparameters. Nucleic acid hybridization will be affected by suchconditions as salt concentration, temperature, solvents, the basecomposition of the hybridizing species, length of the complementaryregions, and the number of nucleotide base mismatches between thehybridizing nucleic acids, as will be readily appreciated by thoseskilled in the art. One having ordinary skill in the art knows how tovary these parameters to achieve a particular stringency ofhybridization.

In general, “stringent hybridization” is performed at about 25° C. belowthe thermal melting point (T_(m)) for the specific DNA hybrid under aparticular set of conditions. “Stringent washing” is performed attemperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions. The T_(m) is thetemperature at which 50% of the target sequence hybridizes to aperfectly matched probe. See Sambrook et al., supra, page 9.51, herebyincorporated by reference. For purposes herein, “high stringencyconditions” are defined for solution phase hybridization as aqueoushybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours,followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes. Itwill be appreciated by the skilled worker that hybridization at 65° C.will occur at different rates depending on a number of factors includingthe length and percent identity of the sequences which are hybridizing.

The nucleic acids (also referred to as polynucleotides) of thisinvention may include both sense and antisense strands of RNA, cDNA,genomic DNA, and synthetic forms and mixed polymers of the above. Theymay be modified chemically or biochemically or may contain non-naturalor derivatized nucleotide bases, as will be readily appreciated by thoseof skill in the art. Such modifications include, for example, labels,methylation, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such asuncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoramidates, carbamates, etc.), charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g.,polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.) Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

The term “mutated” when applied to nucleic acid sequences means thatnucleotides in a nucleic acid sequence may be inserted, deleted orchanged compared to a reference nucleic acid sequence. A singlealteration may be made at a locus (a point mutation) or multiplenucleotides may be inserted, deleted or changed at a single locus. Inaddition, one or more alterations may be made at any number of lociwithin a nucleic acid sequence. A nucleic acid sequence may be mutatedby any method known in the art including but not limited to mutagenesistechniques such as “error-prone PCR” (a process for performing PCR underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product. See, e.g., Leung, D. W., et al., Technique, 1, pp.11-15 (1989) and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2,pp. 28-33 (1992)); and “oligonucleotide-directed mutagenesis” (a processwhich enables the generation of site-specific mutations in any clonedDNA segment of interest. See, e.g., Reidhaar-Olson, J. F. & Sauer, R.T., et al., Science, 241, pp. 53-57 (1988)).

The term “vector” as used herein is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Other vectors include cosmids, bacterial artificialchromosomes (BAC) and yeast artificial chromosomes (YAC). Another typeof vector is a viral vector, wherein additional DNA segments may beligated into the viral genome (discussed in more detail below). Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., vectors having an origin of replication whichfunctions in the host cell). Other vectors can be integrated into thegenome of a host cell upon introduction into the host cell, and arethereby replicated along with the host genome. Moreover, certainpreferred vectors are capable of directing the expression of genes towhich they are operatively linked. Such vectors are referred to hereinas “recombinant expression vectors” (or simply, “expression vectors”).

“Operatively linked” expression control sequences refers to a linkage inwhich the expression control sequence is contiguous with the gene ofinterest to control the gene of interest, as well as expression controlsequences that act in trans or at a distance to control the gene ofinterest.

The term “expression control sequence” as used herein refers topolynucleotide sequences which are necessary to affect the expression ofcoding sequences to which they are operatively linked. Expressioncontrol sequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinant vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism.

The term “peptide” as used herein refers to a short polypeptide, e.g.,one that is typically less than about 50 amino acids long and moretypically less than about 30 amino acids long. The term as used hereinencompasses analogs and mimetics that mimic structural and thusbiological function.

The term “polypeptide” encompasses both naturally-occurring andnon-naturally-occurring proteins, and fragments, mutants, derivativesand analogs thereof. A polypeptide may be monomeric or polymeric.Further, a polypeptide may comprise a number of different domains eachof which has one or more distinct activities.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation (1) isnot associated with naturally associated components that accompany it inits native state, (2) when it exists in a purity not found in nature,where purity can be adjudged with respect to the presence of othercellular material (e.g., is free of other proteins from the samespecies) (3) is expressed by a cell from a different species, or (4)does not occur in nature (e.g., it is a fragment of a polypeptide foundin nature or it includes amino acid analogs or derivatives not found innature or linkages other than standard peptide bonds). Thus, apolypeptide that is chemically synthesized or synthesized in a cellularsystem different from the cell from which it naturally originates willbe “isolated” from its naturally associated components. A polypeptide orprotein may also be rendered substantially free of naturally associatedcomponents by isolation, using protein purification techniques wellknown in the art. As thus defined, “isolated” does not necessarilyrequire that the protein, polypeptide, peptide or oligopeptide sodescribed has been physically removed from its native environment.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion compared toa full-length polypeptide. In a preferred embodiment, the polypeptidefragment is a contiguous sequence in which the amino acid sequence ofthe fragment is identical to the corresponding positions in thenaturally-occurring sequence. Fragments typically are at least 5, 6, 7,8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 aminoacids long, more preferably at least 20 amino acids long, morepreferably at least 25, 30, 35, 40 or 45, amino acids, even morepreferably at least 50 or 60 amino acids long, and even more preferablyat least 70 amino acids long.

A “modified derivative” refers to polypeptides or fragments thereof thatare substantially homologous in primary structural sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate amino acids that are not found in thenative polypeptide. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquitination, labeling, e.g., with radionuclides, and variousenzymatic modifications, as will be readily appreciated by those wellskilled in the art. A variety of methods for labeling polypeptides andof substituents or labels useful for such purposes are well known in theart, and include radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H,ligands which bind to labeled antiligands (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and antiligands whichcan serve as specific binding pair members for a labeled ligand. Thechoice of label depends on the sensitivity required, ease of conjugationwith the primer, stability requirements, and available instrumentation.Methods for labeling polypeptides are well known in the art. See Ausubelet al., 1992, hereby incorporated by reference.

The term “fusion protein” refers to a polypeptide comprising apolypeptide or fragment coupled to heterologous amino acid sequences.Fusion proteins are useful because they can be constructed to containtwo or more desired functional elements from two or more differentproteins. A fusion protein comprises at least 10 contiguous amino acidsfrom a polypeptide of interest, more preferably at least 20 or 30 aminoacids, even more preferably at least 40, 50 or 60 amino acids, yet morepreferably at least 75, 100 or 125 amino acids. Fusion proteins can beproduced recombinantly by constructing a nucleic acid sequence whichencodes the polypeptide or a fragment thereof in frame with a nucleicacid sequence encoding a different protein or peptide and thenexpressing the fusion protein. Alternatively, a fusion protein can beproduced chemically by crosslinking the polypeptide or a fragmentthereof to another protein.

The term “non-peptide analog” refers to a compound with properties thatare analogous to those of a reference polypeptide. A non-peptidecompound may also be termed a “peptide mimetic” or a “peptidomimetic”.See, e.g., Jones, (1992) Amino Acid and Peptide Synthesis, OxfordUniversity Press; Jung, (1997) Combinatorial Peptide and NonpeptideLibraries: A Handbook John Wiley; Bodanszky et al., (1993) PeptideChemistry—A Practical Textbook, Springer Verlag; “Synthetic Peptides: AUsers Guide”, G. A. Grant, Ed, W. H. Freeman and Co., 1992; Evans et al.J. Med. Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986);Veber and Freidinger TINS p. 392 (1985); and references sited in each ofthe above, which are incorporated herein by reference. Such compoundsare often developed with the aid of computerized molecular modeling.Peptide mimetics that are structurally similar to useful peptides of theinvention may be used to produce an equivalent effect and are thereforeenvisioned to be part of the invention.

A “polypeptide mutant” or “mutein” refers to a polypeptide whosesequence contains an insertion, duplication, deletion, rearrangement orsubstitution of one or more amino acids compared to the amino acidsequence of a native or wild type protein. A mutein may have one or moreamino acid point substitutions, in which a single amino acid at aposition has been changed to another amino acid, one or more insertionsand/or deletions, in which one or more amino acids are inserted ordeleted, respectively, in the sequence of the naturally-occurringprotein, and/or truncations of the amino acid sequence at either or boththe amino or carboxy termini. A mutein may have the same but preferablyhas a different biological activity compared to the naturally-occurringprotein.

A mutein has at least 70% overall sequence homology to its wild-typecounterpart. Even more preferred are muteins having 80%, 85% or 90%overall sequence homology to the wild-type protein. In an even morepreferred embodiment, a mutein exhibits 95% sequence identity, even morepreferably 97%, even more preferably 98% and even more preferably 99%,99.5% or 99.9% overall sequence identity. Sequence homology may bemeasured by any common sequence analysis algorithm, such as Gap orBestfit.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinity or enzymatic activity, and (5) confer or modify otherphysicochemical or functional properties of such analogs.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, and other unconventional amino acids may also besuitable components for polypeptides of the present invention. Examplesof unconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, s-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright hand direction is the carboxy-terminal direction, in accordancewith standard usage and convention.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences). In a preferred embodiment, a homologousprotein is one that exhibits 60% sequence homology to the wild typeprotein, more preferred is 70% sequence homology. Even more preferredare homologous proteins that exhibit 80%, 85% or 90% sequence homologyto the wild type protein. In a yet more preferred embodiment, ahomologous protein exhibits 95%, 97%, 98% or 99% sequence identity. Asused herein, homology between two regions of amino acid sequence(especially with respect to predicted structural similarities) isinterpreted as implying similarity in function.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art (see,e.g., Pearson et al., 1994, herein incorporated by reference).

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2) AsparticAcid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild type protein and amutein thereof. See, e.g., GCG Version 6.1.

A preferred algorithm when comparing a inhibitory molecule sequence to adatabase containing a large number of sequences from different organismsis the computer program BLAST (Altschul, S. F. et al. (1990) J. Mol.Biol. 215:403-410; Gish and States (1993) Nature Genet. 3:266-272;Madden, T. L. et al. (1996) Meth. Enzymol. 266:131-141; Altschul, S. F.et al. (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. and Madden, T.L. (1997) Genome Res. 7:649-656), especially blastp or tblastn (Altschulet al., 1997). Preferred parameters for BLASTp are: Expectation value:10 (default); Filter: seg (default); Cost to open a gap: 11 (default);Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Wordsize: 11 (default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using FASTA, a program in GCG Version 6.1.FASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences (Pearson,1990, herein incorporated by reference). For example, percent sequenceidentity between amino acid sequences can be determined using FASTA withits default parameters (a word size of 2 and the PAM250 scoring matrix),as provided in GCG Version 6.1, herein incorporated by reference.

The term “domain” as used herein refers to a structure of a biomoleculethat contributes to a known or suspected function of the biomolecule.Domains may be co-extensive with regions or portions thereof; domainsmay also include distinct, non-contiguous regions of a biomolecule.Examples of protein domains include, but are not limited to, an Igdomain, an extracellular domain, a transmembrane domain, and acytoplasmic domain.

As used herein, the term “molecule” means any compound, including, butnot limited to, a small molecule, peptide, protein, sugar, nucleotide,nucleic acid, lipid, etc., and such a compound can be natural orsynthetic.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ofthe present invention and will be apparent to those of skill in the art.All publications and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

Throughout this specification and its embodiments, the word “comprise”or variations such as “comprises” or “comprising”, will be understood torefer to the inclusion of a stated integer or group of integers but notthe exclusion of any other integer or group of integers.

Nucleic Acid Sequences Encoding Human Endomannosidase Gene

The rat endomannosidase has been cloned (Spiro et al., J. Biol. Chem.272(46):29356-29363 (1997)). Although the rat endomannosidase is theonly cloned member of this family to date, genes and ESTs that showsignificant homology to this ORF, and in particular to the ratendomannosidase catalytic domain, are in databases. By performing aprotein BLAST search using the rat endomannosidase protein sequence(Genbank gi:2642187) we identified two hypothetical human proteins inGenbank having regions of significant homology with the ratendomannosidase sequence (Example 2; FIGS. 3A-C). Combining 5′ and 3′regions of these two hypothetical proteins into one ORF produced aputative sequence of 462 amino acids (FIG. 4) and a predicted molecularmass of 54 kDa. Alignment of this putative human endomannosidasesequence to the known rat sequence indicated that the C-termini of theseproteins are highly conserved but that the N-termini are more varied(FIG. 7). It is likely that the conserved region (i.e., from the motif‘DFQ(K/R)SDRIN’ (SEQ ID NO:27) to the C-terminus), corresponds to thecatalytic domain in each endomannosidase, or at least to a regionessential for activity.

Based on the above-deduced human endomannosidase gene sequence, weconstructed primers and amplified an open reading frame (ORF) from ahuman liver cDNA library by PCR (Example 2). The nucleic acid sequencewhich encodes that ORF is 77.8% identical across its length to thefull-length nucleic acid sequence encoding the rat endomannosidase ORF(sequence pair distances using the Clustal methods with weighted residueweight table). At the amino acid sequence level, the human and ratendomannosidase proteins are predicted to be 76.7% identical overall. Inthe more conserved region noted above (i.e., from the motif‘DFQ(K/R)SDRIN’ (SEQ ID NO:27) to the C-terminus), the proteins are86.6% identical overall. Unlike the rat protein, the predicted humanprotein has a very hydrophobic region at the N-terminus (residues 10 to26) which may be a transmembrane region (FIG. 4, boxed). The humanendomannosidase (unlike the rat protein), is predicted to be a type-IImembrane protein, as are most other higher eukaryotic mannosidases.

We subcloned the human endomannosidase ORF into various vectors,including a yeast integration plasmid (Example 3), to study the effectof its expression on the N-glycosylation pathway of a lower eukaryotichost cell, Pichia pastoris. As described below, engineering the humanmannosidase enzyme into the glycosylation pathway of a host cellsignificantly affects the subsequent glycosylation profile of proteinsproduced in that host cell and its descendants. Preferably, the hostcell is engineered to express a human mannosidase enzyme activity (e.g.,from a catalytic domain) in combination with one or more otherengineered glycosylation activities to make human-like glycoproteins.

Accordingly, the present invention provides isolated nucleic acidmolecules, including but not limited to nucleic acid moleculescomprising or consisting of a full-length nucleic acid sequence encodinghuman endomannosidase. The nucleic acid sequence and the ORF of humanendomannosidase are set forth in FIG. 4 and as SEQ ID NO:1. The encodedamino acid sequence is also set forth in FIG. 4 and in SEQ ID NO:2.

In one embodiment, the invention provides isolated nucleic acidmolecules having a nucleic acid sequence comprising or consisting of awild-type human endomannosidase coding sequence (SEQ ID NO:1); homologs,variants and derivatives thereof; and fragments of any of the above. Inone embodiment, the invention provides a nucleic acid moleculecomprising or consisting of a sequence which is a degenerate variant ofthe wild-type human endomannosidase coding sequence (SEQ ID NO:1). In apreferred embodiment, the invention provides a nucleic acid moleculecomprising or consisting of a sequence which is a variant of the humanendomannosidase coding sequence (SEQ ID NO:1) having at least 65%identity to the wild-type gene. The nucleic acid sequence can preferablyhave at least 70%, 75% or 80% identity to the wild-type humanendomannosidase coding sequence (SEQ ID NO:1) (specifically excluding,however, the rat endomannosidase gene, which is about 78% identicaloverall). Even more preferably, the nucleic acid sequence can have 85%,90%, 95%, 98%, 99%, 99.9%, or higher, identity to the wild-type humanendomannosidase coding sequence (SEQ ID NO:1).

In another embodiment, the nucleic acid molecule of the inventionencodes a polypeptide comprising or consisting of the amino acidsequence of SEQ ID NO:2. Also provided is a nucleic acid moleculeencoding a polypeptide sequence that is at least 65% identical to SEQ IDNO:2 (specifically excluding, however, the rat endomannosidasepolypeptide, which is about 77% identical overall). Typically thenucleic acid molecule of the invention encodes a polypeptide sequence ofat least 70%, 75% or 80% identity to SEQ ID NO:2. Preferably, theencoded polypeptide is at least 85%, 90% or 95% identical to SEQ IDNO:2, and the identity can even more preferably be 98%, 99%, 99.9% oreven higher.

The invention also provides nucleic acid molecules that hybridize understringent conditions to the above-described nucleic acid molecules. Asdefined above, and as is well known in the art, stringent hybridizationsare performed at about 25° C. below the thermal melting point (T_(m))for the specific DNA hybrid under a particular set of conditions, wherethe T_(m) is the temperature at which 50% of the target sequencehybridizes to a perfectly matched probe. Stringent washing is performedat temperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions.

Nucleic acid molecules comprising a fragment of any one of theabove-described nucleic acid sequences are also provided. Thesefragments preferably contain at least 20 contiguous nucleotides. Morepreferably the fragments of the nucleic acid sequences contain at least25, 30, 35, 40, 45 or 50 contiguous nucleotides. Even more preferably,the fragments of the nucleic acid sequences contain at least 60, 70, 80,90, 100 or more contiguous nucleotides. In a further embodiment of theinvention, the nucleic acid sequence is a variant of the fragment havingat least 65% identity to the wild-type gene fragment. The nucleic acidsequence can preferably have at least 70%, 75% or 80% identity to thewild-type gene fragment. Even more preferably, the nucleic acid sequencecan have 85%, 90%, 95%, 98%, 99%, 99.9% or even higher identity to thewild-type gene fragment.

The nucleic acid sequence fragments of the present invention displayutility in a variety of systems and methods. For example, the fragmentsmay be used as probes in various hybridization techniques. Depending onthe method, the target nucleic acid sequences may be either DNA or RNA.The target nucleic acid sequences may be fractionated (e.g., by gelelectrophoresis) prior to the hybridization, or the hybridization may beperformed on samples in situ. One of skill in the art will appreciatethat nucleic acid probes of known sequence find utility in determiningchromosomal structure (e.g., by Southern blotting) and in measuring geneexpression (e.g., by Northern blotting). In such experiments, thesequence fragments are preferably detectably labeled, so that theirspecific hydridization to target sequences can be detected andoptionally quantified. One of skill in the art will appreciate that thenucleic acid fragments of the present invention may be used in a widevariety of blotting techniques not specifically described herein.

It should also be appreciated that the nucleic acid sequence fragmentsdisclosed herein also find utility as probes when immobilized onmicroarrays. Methods for creating microarrays by deposition and fixationof nucleic acids onto support substrates are well known in the art.Reviewed in DNA Microarrays: A Practical Approach (Practical ApproachSeries), Schena (ed.), Oxford University Press (1999) (ISBN:0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray BiochipTools and Technology, Schena (ed.), Eaton PublishingCompany/BioTechniques Books Division (2000) (ISBN: 1881299376), thedisclosures of which are incorporated herein by reference in theirentireties. Analysis of, for example, gene expression using microarrayscomprising nucleic acid sequence fragments, such as the nucleic acidsequence fragments disclosed herein, is a well-established utility forsequence fragments in the field of cell and molecular biology. Otheruses for sequence fragments immobilized on microarrays are described inGerhold et al., Trends Biochem. Sci. 24:168-173 (1999) and Zweiger,Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A PracticalApproach (Practical Approach Series), Schena (ed.), Oxford UniversityPress (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999);Microarray Biochip Tools and Technology, Schena (ed.), Eaton PublishingCompany/BioTechniques Books Division (2000) (ISBN: 1881299376), thedisclosures of each of which is incorporated herein by reference in itsentirety. In another embodiment, isolated nucleic acid moleculesencoding a polypeptide having endomannosidase activity are provided. Asis well known in the art, enzyme activities can be measured in variousways. Alternatively, the activity of the enzyme can be followed usingchromatographic techniques, such as by high performance liquidchromatography. Chung and Sloan, J. Chromatogr. 371:71-81 (1986). Othermethods and techniques may also be suitable for the measurement ofenzyme activity, as would be known by one of skill in the art.

In another embodiment, the nucleic acid molecule of the inventionencodes a polypeptide having the amino acid sequence of SEQ ID NO:2. Thenucleic acid sequence of the invention encodes a polypeptide having atleast 77% identity to the wild-type rat endomannosidase gene (GenbankAF023657). In another embodiment, the nucleic acid sequence has at least87% identity to the wild-type rat endomannosidase catalytic domain. Inan even more preferred embodiment, the nucleic acid sequence can have90%, 95%, 98%, 99%, 99.9% or even higher identity to the wild-type ratendomannosidase gene.

Polypeptides encoded by the nucleic acids of the invention, especiallypeptides having a biological (e.g., catalytic or other) and/orimmunological activity, are also provided by the invention.

Nucleic Acid Sequences Encoding Mouse Endomannosidase Gene

The mouse endomannosidase gene is cloned by designing primers thatcomplement the putative homologous regions between the mouse and humanendomannosidase genes and PCR amplifying to generate a probe which canbe used to pull out a full-length cDNA encoding mouse endomannosidase(Example 2). The nucleotide and predicted amino acid sequence of themouse endomannosidase open reading frame (ORF) is set forth in FIG. 6and as SEQ ID NOs:3 and 4, respectively.

The mouse ORF shows substantial homology to the known ratendomannosidase and the human liver endomannosidase of the presentinvention (FIG. 7). Specifically, the nucleic acid sequence whichencodes the mouse endomannosidase ORF is 86.0% and 84.2% identicalacross its length to the full-length nucleic acid sequence encoding therat and the human endomannosidase ORFs, respectively (sequence pairdistances using the Clustal methods with weighted residue weight table).At the amino acid sequence level, the mouse and rat endomannosidaseproteins are predicted to be 82.3% identical, and the mouse and humanendomannosidase proteins are predicted to be 84.9% identical overall. Inthe more conserved region noted above (i.e., from the motif‘DFQ(K/R)SDRIN’ (SEQ ID NO:27) to the C-terminus), the mouse and ratproteins are 92.3% identical, and the mouse and human proteins are 86.1%identical, overall.

Accordingly, the present invention further provides isolated nucleicacid molecules and variants thereof encoding the mouse endomannosidase.In one embodiment, the invention provides an isolated nucleic acidmolecule having a nucleic acid sequence comprising or consisting of thegene encoding the mouse endomannosidase (SEQ ID NO:3), homologs,variants and derivatives thereof.

Accordingly, the present invention provides isolated nucleic acidmolecules, including but not limited to nucleic acid moleculescomprising or consisting of a full-length nucleic acid sequence encodingmouse endomannosidase. The nucleic acid sequence and the ORF of mouseendomannosidase are set forth in FIG. 6 and as SEQ ID NO:3. The encodedamino acid sequence is also set forth in FIG. 6 and in SEQ ID NO:4.

In one embodiment, the invention provides isolated nucleic acidmolecules having a nucleic acid sequence comprising or consisting of awild-type mouse endomannosidase coding sequence (SEQ ID NO:3); homologs,variants and derivatives thereof; and fragments of any of the above. Inone embodiment, the invention provides a nucleic acid moleculecomprising or consisting of a sequence which is a degenerate variant ofthe wild-type mouse endomannosidase coding sequence (SEQ ID NO:3). In apreferred embodiment, the invention provides a nucleic acid moleculecomprising or consisting of a sequence which is a variant of the mouseendomannosidase coding sequence (SEQ ID NO:3) having at least 65%identity to the wild-type gene. The nucleic acid sequence can preferablyhave at least 70%, 75%, 80% or 85% identity to the wild-type humanendomannosidase coding sequence (SEQ ID NO:3) (specifically excluding,however, the rat endomannosidase gene, which is about 86% identicaloverall). Even more preferably, the nucleic acid sequence can have 90%,95%, 98%, 99%, 99.9%, or higher, identity to the wild-type mouseendomannosidase coding sequence (SEQ ID NO:3).

In another embodiment, the nucleic acid molecule of the inventionencodes a polypeptide comprising or consisting of the amino acidsequence of SEQ ID NO:4. Also provided is a nucleic acid moleculeencoding a polypeptide sequence that is at least 65% identical to SEQ IDNO:4 (specifically excluding, however, the rat endomannosidasepolypeptide, which is about 82% identical overall). Typically thenucleic acid molecule of the invention encodes a polypeptide sequence ofat least 70%, 75% or 80% identity to SEQ ID NO:4. Preferably, theencoded polypeptide is at least 85%, 90% or 95% identical to SEQ IDNO:4, and the identity can even more preferably be 98%, 99%, 99.9% oreven higher.

The invention also provides nucleic acid molecules that hybridize understringent conditions to the above-described nucleic acid molecules. Asdefined above, and as is well known in the art, stringent hybridizationsare performed at about 25° C. below the thermal melting point (T_(m))for the specific DNA hybrid under a particular set of conditions, wherethe T_(m) is the temperature at which 50% of the target sequencehybridizes to a perfectly matched probe. Stringent washing is performedat temperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions.

Nucleic acid molecules comprising a fragment of any one of theabove-described nucleic acid sequences are also provided. Thesefragments preferably contain at least 20 contiguous nucleotides. Morepreferably the fragments of the nucleic acid sequences contain at least25, 30, 35, 40, 45 or 50 contiguous nucleotides. Even more preferably,the fragments of the nucleic acid sequences contain at least 60, 70, 80,90, 100 or more contiguous nucleotides. In a further embodiment of theinvention, the nucleic acid sequence is a variant of the fragment havingat least 65% identity to the wild-type gene fragment. The nucleic acidsequence can preferably have at least 70%, 75% or 80% identity to thewild-type gene fragment. Even more preferably, the nucleic acid sequencecan have 85%, 90%, 95%, 98%, 99%, 99.9% or even higher identity to thewild-type gene fragment.

In another embodiment, the nucleic acid molecule of the inventionencodes a polypeptide comprising or consisting of the amino acidsequence of SEQ ID NO:4. Also provided is a nucleic acid moleculeencoding a polypeptide sequence that is at least 65% identical to SEQ IDNO:4 (specifically excluding, however, the rat endomannosidasepolypeptide, which is about 82% identical overall). Typically thenucleic acid molecule of the invention encodes a polypeptide sequence ofat least 70%, 75% or 80% identity to SEQ ID NO:4. Preferably, theencoded polypeptide is at least 85%, 90% or 95% identical to SEQ IDNO:4, and the identity can even more preferably be 98%, 99%, 99.9% oreven higher.

In a preferred embodiment, the nucleic acid molecule of the inventionencodes a polypeptide having at least 83% identity to the wild-type ratendomannosidase gene (Genbank AF023657). In another embodiment, thenucleic acid sequence encoding an amino acid sequence has at least 93%identity to the wild-type rat endomannosidase catalytic domain. In aneven more preferred embodiment, the nucleic acid sequence can have 94%,95%, 98%, 99%, 99.9% or even higher identity to the wild-type ratendomannosidase gene.

Polypeptides encoded by the nucleic acids of the invention, especiallypeptides having a biological (e.g., catalytic or other) and/orimmunological activity, are also provided by the invention.

Characterization of Encoded Endomannosidase Products

The human liver endomannosidase and the putative mouse endomannosidaseare the second and third members of a newly developing family ofglycosidic enzymes, with the rat endomannosidase enzyme being the firstsuch member. Sequence comparison of the human, mouse and rat ORFs (FIG.7) demonstrates high homology from the motif ‘DFQ(K/R)SDRI’ (residues1-8 of SEQ ID NO:27) to the C-termini of the sequences suggesting thatthis region encodes an essential fragment of the protein, andpotentially, the catalytic domain. In contrast, the lower homologywithin the N-termini of the proteins demonstrates evolutionarydivergence. Like the majority of glycosidases and glycosyltransferases,the mouse and human enzymes have a hydrophobic region indicative of atransmembrane domain. Such a domain would facilitate the orientation andlocalization of the enzyme in the secretory pathway. In contrast, therat endomannosidase does not have a transmembrane domain but does have aglycine residue at position 2 (Spiro 1997, supra). This penultimateglycine residue has the potential to be myristoylated which in turnprovides a mechanism for membrane localization (Boutin, Cell Signal 9:15-35 (1997)). Alternatively, myristoylation may not be the means of ratendomannosidase localization to the Golgi (Zuber 2000,supra)—protein-protein interactions may be the determining mechanism.

Like the rat endomannosidase, both the human and mouse isoforms arepredicted to localize to the Golgi based on the activity of this classof proteins. Traditionally, the removal of glucose from N-glycans wasthought to occur in the ER by glucosidases I and II. However, thecharacterization of endomannosidase and its localization to the cis andmedial cisternae of the Golgi demonstrates that glucose trimming doesoccur subsequent to glucosidase localization (Roth et al. Biochimie 85:287-294 (2003)).

The specific role that endomannosidase fulfills is currently uncertain.Affinity-purification of rat endomannosidase demonstrated theco-purification with calreticulin suggesting its role in the qualitycontrol of N-glycosylation (Spiro et al., J. Biol. Chem. 271:11588-11594 (1996)). Alternatively, endomannosidase may provide the cellwith the ability to recover and properly mature glucosylated structuresthat have by-passed glucosidase trimming. Thus, removing theglucose-α1,3-mannose dimer from a glucosylated high mannose structurepresents a substrate for the resident Golgi glycosidic andglycosyltransferase enzymes, enabling the maturation of the N-glycans.

We analyzed the tissue distribution of human endomannosidase and, likethe rat isoform (Spiro (1997)), it was widespread in the tissuesexamined (FIG. 8) (Example 6). The liver and kidney demonstrated highexpression levels but the pattern in the remainder of the tissues wassignificantly different. Interestingly, in contrast to the humanendomannosidase, the rat isoform shows high expression levels in boththe brain and lung (Spiro (1997)). The widespread expression of bothisoforms of this enzyme in rat and human suggests that endomannosidasemay play a house-keeping role in the processing of N-glycans.

Expression in P. pastoris of the human endomannosidase of the inventionconfirms that the isolated ORF has activity. Interestingly, the ratisoform, though highly homologous at the nucleotide and protein levels,is expressed at levels at least five-fold higher than the human proteinas seen on Western Blots (FIG. 9). It is possible that rat enzyme isinherently more stable during expression or in the culture medium.

Both recombinantly expressed endomannosidase enzymes were processed attheir C-termini. In the case of the human enzyme, C-terminal processingappeared to be complete (based on apparent total conversion of the 59kDa band to the 54 kDa form, presumably due to the lower expressionlevel). In contrast, though the majority of the rat isoform was the 54kDa form, some of the 59 kDa band remained (Example 7). Likewise, whenthe rat endomannosidase was expressed in Escherichia coli, the proteinwas proteolytically processed at the C-terminus over time (Spiro 1997,supra). Furthermore, affinity chromatographic purification of the ratisoform from rat liver demonstrated the presence of two forms, 56 and 60kDa (Hiraizumi et al., J. Biol. Chem. 269: 4697-4700 (1994)). Together,these data indicate that both the human and rat endomannosidase proteinsare susceptible to proteolytic processing. Based on the similar sizes ofthe two enzymes following proteolysis, the cleavage site is likely thesame. Whether the cleavage site in the bacterial, yeast and mammaliansystems is the same remains to be determined. Further characterizationof the endomannosidase shows an optimal activity at about pH 6.2(Example 9) and a temperature optimum of about 37° C. (Example 9).

The isolation and characterization of the human endomannosidase and theidentification of the mouse homologue expands this family ofglycosidases from a solitary member consisting of the rat isoform. Thisin turn has allowed us to characterize further this family of proteins.Indeed, this has allowed us to demonstrate that, while the C-terminalsequences of these proteins are highly conserved, variations in theN-terminal architecture occur. A previously reported phylogenetic surveyof endomannosidase indicated that this protein has emerged only recentlyduring evolution and is restricted to members of the chordate phylum,which includes mammals, birds, reptiles, amphibians and bony fish, withthe only exception being that it has also been identified in Mollusca(Dairaku and Spiro, Glycobiology 7: 579-586 (1997)). Therefore, theisolation of more diversified members of this family of proteins willexpectedly demonstrate further variations in endomannosidase structureand, potentially, activity.

Utility of Endomannosidase Expression

The human and mouse endomannosidase enzymes or catalytic domains (andnucleic acid molecules of the invention encoding such activities) willeach be useful, e.g., for modifying certain glycosylation structures, inparticular, for hydrolyzing a composition comprising at least oneglucose residue and one mannose residue on a glucosylated glycanstructure (FIG. 1 and FIG. 2). In one embodiment, the encoded enzymecatalyzes the cleavage of a di- tri-, or tetra-saccharide compositioncomprising at least one glucose residue and one mannose residue ofglucosylated glycan precursors (FIG. 1). In another embodiment, theencoded enzyme also modifies a number of glucosylated structures,including Glc₁₋₃Man₉₋₅GlcNAc₂ (FIG. 2). One or more nucleic acids and/orpolypeptides of the invention are introduced into a host cell of choiceto modify the glycoproteins produced by that host cell.

Cellular Targeting of Endomannosidase In Vivo

Although glucosidases act upon high mannan glycans in the ER, somemannans escape the ER without proper modification and, thus, mannanswith undesired glycosylations move through the secretory pathway.Previous studies suggest that in higher eukaryotes a fraction ofglucosylated mannose structures does bypass the quality control of theER, and that endomannosidase is present in the subsequent compartment torecover this fraction. Accordingly, in a feature of the presentinvention, the endomannosidase modifies the glucosylated mannosestructures that have bypassed the ER. In a preferred embodiment, theendomannosidase enzyme encoded by the nucleic acid of the presentinvention is localized in the Golgi, trans Golgi network, transportvesicles or the ER. The enzymes are involved in the trimming ofglucosylated high mannan glycans in yeast. For example, the glucosylatedstructure GlcMan₉GlcNAc₂, which has bypassed the ER glucosidase I and IIenzymes, is modified by the endomannosidase in which at least aglucose-mannose residue is hydrolyzed producing Man₈GlcNAc₂. Theendomannosidase enzymes of the present invention act as a qualitycontrol step in the Golgi, recovering the glucosylated high mannanglycans and removing a composition comprising at least one glucoseresidue and one mannose residue.

Combinatorial Nucleic Acid Library Encoding Endomannosidase CatalyticDomains

In another aspect of the invention, one or more chimeric nucleic acidmolecules encoding novel endomannosidase proteins is constructed byforming a fusion protein between an endomannosidase enzyme and acellular targeting signal peptide, e.g., by the in-frame ligation of aDNA fragment encoding a cellular targeting signal peptide with a DNAfragment encoding an endomannosidase enzyme or catalytically activefragment thereof. Preferably, one or more fusion proteins are made inthe context of an endomannosidase combinatorial DNA library. Seegenerally WO 02/00879 and the publication of U.S. application Ser. No.10/371,877 (filed Feb. 20, 2003); each of which is incorporated hereinby reference in nits entirety. The endomannosidase DNA library comprisesa wide variety of fusion constructs, which are expressed in a host cellof interest, e.g., by using an integration plasmid such as the pRCD259(Example 5).

Targeting Peptide Sequence Sub-Libraries

Another useful sub-library includes nucleic acid sequences encodingtargeting signal peptides that result in localization of a protein to aparticular location within the ER, Golgi, or trans Golgi network. Thesetargeting peptides may be selected from the host organism to beengineered as well as from other related or unrelated organisms.Generally such sequences fall into three categories: (1) N-terminalsequences encoding a cytosolic tail (ct), a transmembrane domain (tmd)and part or all of a stem region (sr), which together or individuallyanchor proteins to the inner (lumenal) membrane of the Golgi; (2)retrieval signals which are generally found at the C-terminus such asthe HDEL (SEQ ID NO:28) or KDEL (SEQ ID NO:29) tetrapeptide; and (3)membrane spanning regions from various proteins, e.g., nucleotide sugartransporters, which are known to localize in the Golgi.

In the first case, where the targeting peptide consists of variouselements (cytosolic tail (ct), transmembrane domain (tmd) and stemregion (sr)), the library is designed such that the ct, the tmd andvarious parts of the stem region are represented. Accordingly, apreferred embodiment of the sub-library of targeting peptide sequencesincludes ct, tmd, and/or sr sequences from membrane-bound proteins ofthe ER or Golgi. In some cases it may be desirable to provide thesub-library with varying lengths of sr sequence. This may beaccomplished by PCR using primers that bind to the 5′ end of the DNAencoding the cytosolic region and employing a series of opposing primersthat bind to various parts of the stem region.

Still other useful sources of targeting peptide sequences includeretrieval signal peptides, e.g. the tetrapeptides HDEL (SEQ ID NO:28;also shown in column 1 of Table 1) or KDEL (SEQ ID NO:29), which aretypically found at the C-terminus of proteins that are transportedretrograde into the ER or Golgi. Still other sources of targetingpeptide sequences include (a) type II membrane proteins, (b) the enzymeswith optimum pH, (c) membrane spanning nucleotide sugar transportersthat are localized in the Golgi, and (d) sequences referenced in Table1.

TABLE 1 Sources of useful compartmental targeting sequences Gene orLocation of Gene Sequence Organism Function Product MNSI A. nidulansα-1,2-mannosidase ER MNSI A. niger α-1,2-mannosidase ER MNSI S.cerevisiae α-1,2-mannosidase ER GLSI S. cerevisiae glucosidase ER GLSIA. niger glucosidase ER GLSI A. nidulans glucosidase ER HDEL Universalin fungi retrieval signal ER at C-terminus SEC12 S. cerevisiae COPIIvesicle protein ER/Golgi SEC12 A. niger COPII vesicle protein ER/GolgiOCH1 S. cerevisiae 1,6-mannosyltransferase Golgi (cis) OCH1 P. pastoris1,6-mannosyltransferase Golgi (cis) MNN9 S. cerevisiae1,6-mannosyltransferase Golgi complex MNN9 A. niger undetermined GolgiVAN1 S. cerevisiae undetermined Golgi VAN1 A. niger undetermined GolgiANP1 S. cerevisiae undetermined Golgi HOCI S. cerevisiae undeterminedGolgi MNN10 S. cerevisiae undetermined Golgi MNN10 A. niger undeterminedGolgi MNN11 S. cerevisiae undetermined Golgi (cis) MNN11 A. nigerundetermined Golgi (cis) MNT1 S. cerevisiae 1,2-mannosyltransferaseGolgi (cis, medial KTR1 P. pastoris undetermined Golgi (medial) KRE2 P.pastoris undetermined Golgi (medial) KTR3 P. pastoris undetermined Golgi(medial) MNN2 S. cerevisiae 1,2-mannosyltransferase Golgi (medial) KTR1S. cerevisiae undetermined Golgi (medial) KTR2 S. cerevisiaeundetermined Golgi (medial) MNN1 S. cerevisiae 1,3-mannosyltransferaseGolgi (trans) MNN6 S. cerevisiae Phosphomannosyltransferase Golgi(trans) 2,6 ST H. sapiens 2,6-sialyltransferase trans Golgi networkUDP-Gal T S. pombe UDP-Gal transporter Golgi

Endomannosidase Fusion Constructs

A representative example of an endomannosidase fusion construct derivedfrom a combinatorial DNA library of the invention inserted into aplasmid is pSH280, which comprises a truncated Saccharomyces MNN11(m)targeting peptide (1-303 nucleotides of MNN11 from SwissProt P46985),constructed from primers SEQ ID NO: 5 and SEQ ID NO: 6, ligated in-frameto a 48 N-terminal amino acid deletion of a rat endo-α1,2-mannosidase(Genbank AF 023657). The nomenclature used herein, thus, refers to thetargeting peptide/catalytic domain region of a glycosylation enzyme asSaccharomyces MNN11(m)/rat endomannosidase Δ48. The encoded fusionprotein localizes in the Golgi by means of the MNN11 targeting peptidesequence while retaining its endomannosidase catalytic domain activityand is capable of producing unglucosylated N-glycans such as Man₄GlcNAc₂in a lower eukaryote. The glycan profile from a reporter glycoprotein K3expressed in a strain of P. pastoris RDP25 (och1 alg3) transformed withpSH280 exhibits a peak, among others, at 1099 m/z [c] corresponding tothe mass of Man₄GlcNAc₂ and 1424 m/z [a] corresponding to the mass ofhexose 6 (FIG. 10B; see Examples 11 and 12). This new P. pastorisstrain, designated as YSH97, shows greater than about 95%endomannosidase activity evidenced by the extent to which theglucosylated hexose 6 structure is removed from the reporterglycoprotein.

The structure of hexose 6 [a] expressed in a host cell (e.g., P.pastoris RDP25) comprises a mixture of glycans comprising GlcMan₅GlcNAc₂and Man₆GlcNAc₂ and its isomers (FIG. 10A). By introduction andexpression of the endomannosidase of the present invention in a hostcell, a composition comprising at least one glucose residue and mannoseresidue is removed from the hexose 6 structure (FIG. 10B). Theglucosylated structure GlcMan₅GlcNAc₂ is readily converted toMan₄GlcNAc₂, which is then subsequently converted to Man₃GlcNAc₂ withα1,2-mannosidase in vitro digestion. The hexose 6 species comprising theglucosylated mannans is not cleaved by α1,2-mannosidase. The predominantpeak corresponding to the structure Man₃GlcNAc₂ [b] (FIG. 10C) shownafter the α1,2-mannosidase digestion confirms the apparent removal ofthe glucose-mannose dimer from GlcMan₅GlcNAc₂ exposing a terminalManα1,2 on Man₄GlcNAc₂ for hydrolysis producing Man₃GlcNAc₂.

The other species of hexose 6: Man₆GlcNAc₂ is not readily affected bythe endomannosidase of the present invention and accordingly, iscontemplated as un-glucosylated structures. A skilled artisan wouldappreciate that this species of hexose 6: Man₆GlcNAc₂ comprises Manα1,2additions, which is evidenced by the subsequent α1,2-mannosidase invitro digestion producing Man₃GlcNAc₂ (FIG. 10C).

Another example of an endomannosidase fusion construct derived from acombinatorial DNA library of the invention inserted into a plasmid ispSH279, which is a truncated Saccharomyces VAN1(s) targeting peptide(1-279 nucleotides of VAN1 from SwissProt P23642) constructed fromprimers SEQ ID NO: 7 and SEQ ID NO: 8, ligated in-frame to a 48N-terminal amino acid deletion of a rat endo-α1,2-mannosidase (GenbankAF 023657). The nomenclature used herein, thus, refers to the targetingpeptide/catalytic domain region of a glycosylation enzyme asSaccharomyces VAN1(s)/rat endomannosidase Δ48. The encoded fusionprotein localizes in the Golgi by means of the VAN1 targeting peptidesequence while retaining its endomannosidase catalytic domain activityand is capable of producing N-glycans having a Man₄GlcNAc₂ structure inP. pastoris (RDP25). The glycan profile from a reporter glycoprotein K3expressed in a strain of P. pastoris RDP-25 (och1 alg3) transformed withpSH279 exhibits a peak, among others, at 1116 m/z [c] corresponding tothe mass of Man₄GlcNAc₂ and 1441 m/z [a] corresponding to the mass ofhexose 6 (FIG. 11; examples 11 and 12). FIG. 11B shows a residual hexose6 [a] peak indicating only partial activity of the endomannosidase. Thisstrain, designated as YSH96, shows greater than about 40%endomannosidase activity, evidenced by the extent to which theglucosylated hexose 6 structure is removed from the reporterglycoprotein.

The structure of hexose 6 [a] expressed in a host cell (e.g., P.pastoris RDP25) comprises a mixture of glycans comprising GlcMan₅GlcNAc₂and Man₆GlcNAc₂ and its isomers (FIG. 11A). By introduction andexpression of the endomannosidase of the present invention in a hostcell, a composition comprising at least one glucose residue and mannoseresidue is removed from the hexose 6 structure (FIG. 11B). Theglucosylated structure GlcMan₅GlcNAc₂ is readily converted toMan₄GlcNAc₂, which is then subsequently converted to Man₃GlcNAc₂ withα1,2-mannosidase in vitro digestion. The hexose 6 species comprising theglucosylated mannans is not cleaved by α1,2-mannosidase. The predominantpeak corresponding to the structure Man₃GlcNAc₂ [b] (FIG. 11C) shownafter the α1,2-mannosidase digestion confirms the apparent removal ofthe glucose-mannose dimer from GlcMan₅GlcNAc₂ exposing a terminalManα1,2 on Man₄GlcNAc₂ for hydrolysis producing Man₃GlcNAc₂.

The other species of hexose 6: Man₆GlcNAc₂ is not readily affected bythe endomannosidase of the present invention and accordingly, iscontemplated as un-glucosylated structures. A skilled artisan wouldappreciate that this species of hexose 6: Man₆GlcNAc₂ comprises Manα1,2additions, which is evidenced by the subsequent α1,2-mannosidase invitro digestion producing Man₃GlcNAc₂ (FIG. 11C).

Additionally, an example of an endomannosidase fusion construct insertedinto a plasmid that does not show apparent catalytic activity derivedfrom a combinatorial DNA library of the invention is pSH278, which atruncated Saccharomyces GLS1(s) targeting peptide (1-102 nucleotides ofGLS1 from SwissProt P53008) constructed from primers SEQ ID NO: 9 andSEQ ID NO: 10, ligated in-frame to a 48 N-terminal amino acid deletionof a rat endo-α1,2-mannosidase (Genbank AF 023657). The nomenclatureused herein, thus, refers to the targeting peptide/catalytic domainregion of a glycosylation enzyme as Saccharomyces GLS1(s)/ratendomannosidase A48. The glycan profile from a reporter glycoprotein K3expressed in a strain of a P. pastoris RDP-25 (och1 alg3) transformedwith pSH278 exhibits, a peak, among others, at 1439 m/z (K⁺ adduct) [c]and a peak at 1422 m/z (Na⁺ adduct) corresponding to the mass of hexose6 [a](FIG. 12; examples 11 and 12). This strain, designated as YSH95,shows less than about 10% endomannosidase activity as evidenced by theextent to which the glucosylated hexose 6 structure is removed from thereporter glycoprotein.

Unlike the previous two glycan profiles shown in FIGS. 10 and 11, theendomannosidase construct pSH278 expressed in P. pastoris RDP25 showsrelatively low endomannosidase activity (FIG. 12). Subsequent digestionwith α1,2 mannosidase, however, reveals a peak corresponding to the massof Man₃GlcNAc₂ [b]. A skilled artisan would appreciate that the hexose 6species comprising Man₆GlcNAc₂ have been converted to Man₃GlcNAc₂ byintroduction of α1,2 mannosidase whereas the other hexose 6 speciescomprising GlcMan₅GlcNAc₂ are still present, which, in effect, are stillglucosylated.

By creating a combinatorial DNA library of these and other suchendomannosidase fusion constructs according to the invention, a skilledartisan may distinguish and select those constructs having optimalintracellular endomannosidase trimming activity from those havingrelatively low or no activity. Methods using combinatorial DNA librariesof the invention are advantageous because only a select fewendomannosidase fusion constructs may produce a particularly desiredN-glycan in vivo. In addition, endomannosidase trimming activity may bespecific to a particular protein of interest. Thus, it is to be furtherunderstood that not all targeting peptide/mannosidase catalytic domainfusion constructs may function equally well to produce the properglycosylation on a glycoprotein of interest. Accordingly, a protein ofinterest may be introduced into a host cell transformed with acombinatorial DNA library to identify one or more fusion constructswhich express a mannosidase activity optimal for the protein ofinterest. One skilled in the art will be able to produce and selectoptimal fusion construct(s) using the combinatorial DNA library approachdescribed herein.

It is apparent, moreover, that other such fusion constructs exhibitinglocalized active endomannosidase catalytic domains may be made usingtechniques such as those exemplified in WO 02/00879 and describedherein. It will be a matter of routine experimentation for one skilledin the art to make and use the combinatorial DNA library of the presentinvention to optimize non-glucosylated N-glycans (for exampleMan₄GlcNAc₂) production from a library of fusion constructs in aparticular expression vector introduced into a particular host cell.

Recombinant Expression of Genes Encoding Endomannosidase

Another feature of the invention is the recombinant expression of thenucleic acid sequences encoding the endomannosidase. The nucleic acidsequences are operatively linked to an expression control sequence in anappropriate expression vector and transformed in an appropriate hostcell (Example 3). A wide variety of suitable vectors readily availablein the art are used to express the fusion constructs of the presentinvention in a variety of host cells. The vectors pSH278, pSH279 andpSH280 (Example 4) are a select few examples described herein suitablefor expression of endomannosidase activity in a lower eukarote, Pichiapastoris. It is to be understood that a wide variety of vectors suitablefor expression of endomannosidase activity in a selected host cell areencompassed within the present invention.

In one aspect of the invention, a lower eukaryotic host cell producingglucosylated high mannose structures is modified by introduction andexpression of the endomannosidase of the present invention. For example,a host cell P. pastoris RDP25 (och1 alg3) producing hexose 6 is modifiedby introduction and expression of the endomannosidase of the presentinvention. The host cell of the present invention produces a modifiedglycan converting GlcMan₅GlcNAc₂ to Man₄GlcNAc₂. Accordingly, in oneembodiment, a lower eukaryotic host cell expressing the endomannosidaseof the present invention catalyzes the removal of a molecule comprisingat least one glucose residue and a mannose residue.

The activity of the recombinant nucleic acid molecules encoding theendomannosidase of the invention are described herein. Varied expressionlevels are quantified by the conversion of a glucosylated glycanGlcMan₅GlcNAc₂ to a deglucosylated glycan Man₄GlcNAc₂. In oneembodiment, the conversion of GlcMan₅GlcNAc₂ to Man₄GlcNAc₂ is partial(FIG. 10, 11).

In another embodiment, the conversion of GlcMan₅GlcNAc₂ to Man₄GlcNAc₂is complete. In a preferred embodiment, at least 30% of GlcMan₅GlcNAc₂is converted to Man₄GlcNAc₂. In a more preferred embodiment, at least60% of GlcMan₅GlcNAc₂ is converted to Man₄GlcNAc₂. In an even morepreferred embodiment, at least 90% of GlcMan₅GlcNAc₂ is converted toMan₄GlcNAc₂. Furthermore, it is contemplated that other glucosecontaining glycans are removed by the endomannosidase of the presentinvention. For example, the endomannosidase of the present inventionfurther comprises the activity of truncating a glycanGlc₁₋₃Man₉₋₅GlcNAc₂ to Man₈₋₄GlcNAc₂.

Additionally, a gene encoding a catalytically active endomannosidase isexpressed in a lower eukaryotic host cell (e.g. Pichia pastoris)modifying the glycosylation on a protein of interest. In one embodiment,the endomannosidase of the present invention modifies glucosylatedN-linked oligosaccharides on a protein of interest. The resultingprotein produces a more human-like glycoprotein. A lower eukaryotic hostcell modified by the endomannosidase of the invention produces aMan₈₋₄GlcNAc₂ glycoform from a glucosylated glycoform on a protein ofinterest (FIG. 2). For example, a strain of P. pastoris modified by theendomannosidae of the invention produces a Man₄GlcNAc₂ glycoform anddecreased moiety of the glucosylated hexose 6 glycoform on a protein ofinterest (FIG. 10B). Subsequent α1,2-mannosidase digestion of theMan₄GlcNAc₂ glycoform results in a trimannosyl core (FIG. 10C).Accordingly, the present invention provides a catalytically activeendomannosidase in a lower eukaryotic host cell that converts aglucosylated glycoform to a desired glycoform on a therapeutic proteinof interest.

Therapeutic proteins are typically administered by injection, orally,pulmonary, or other means. Examples of suitable target glycoproteinswhich may be produced according to the invention include, withoutlimitation: erythropoietin, cytokines such as interferon-α,interferon-β, interferon-γ, interferon-ω, and granulocyte-CSF,coagulation factors such as factor VIII, factor IX, and human protein C,soluble IgE receptor α-chain, IgG, IgG fragments, IgM, interleukins,urokinase, chymase, and urea trypsin inhibitor, IGF-binding protein,epidermal growth factor, growth hormone-releasing factor, annexin Vfusion protein, angiostatin, vascular endothelial growth factor-2,myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1-antitrypsinand α-feto proteins.

Promoters

In another aspect of the invention, the rat liver endomannosidase(Genbank gi:2642186), the human endomannosidase (Genbank gi:20547442) orthe mouse mannosidase (Genbank AK030141) is cloned into a yeastintegration plasmid under the control of a constitutive promoter tooptimize the amount of endomannosidase activity while restrictingadverse effects on the cell. This involves altering promoter strengthand optionally includes using an inducible promoter to better controlthe expression of these proteins.

In addition to expressing the wild-type endomannosidase, modified formsof the endomannosidase are expressed to enhance cellular localizationand activity. Varying lengths of the catalytic domain of endomannosidaseis fused to endogenous yeast targeting regions as described in WO02/00879. The catalytically active fragment encoding the endomannosidasegenes are cloned into a yeast integration plasmid under the control of aconstitutive promoter. This involves altering the promoter strength andmay include using an inducible promoter to better control the expressionof these proteins. Furthermore, to increase enzyme activity, the proteinis mutated to generate new characteristics. The skilled artisanrecognizes the routine modifications of the procedures disclosed hereinmay provide improved results in the production of unglucosylatedglycoprotein of interest.

Codon Optimization

It is also contemplated that the nucleic acids of the present inventionmay be codon optimized resulting in one or more changes in the primaryamino acid sequence, such as a conservative amino acid substitution,addition, deletion or combination thereof.

Secreted Endomannosidase

In another feature of the invention, a soluble secreted endomannosidaseis expressed in a host cell. In a preferred embodiment, a soluble mouseor human endomannosidase is recombinantly expressed. A solubleendomannosidase lacks cellular localization signal that normallylocalizes to the Golgi apparatus or bind to the cell membrane.Expression of the catalytic domain of the endomannosidase to produce asoluble recombinant enzyme, which lacks the transmembrane domain, can befused in-frame to a second domain or a tag that facilitates itspurification. The secreted rat and human endomannosidase of the presentinvention from P. pastoris is shown in FIG. 9 (Example 8).

Expressed endomannosidase is particularly useful for in vitromodification of glucosylated glycan structures. In a more preferredembodiment, the recombinant endomannosidase is used to produceunglucosylated glycan intermediates in large scale glycoproteinproduction. FIG. 13 shows the activity of the rat (FIG. 13B) and human(FIG. 13C) endomannosidase that have cleaved the glucose-α1,3-mannosedimer on the glycan intermediate GlcMan₅GlcNAc₂ converting it toMan₄GlcNAc₂. (See FIG. 14). Accordingly, the endomannosidase of thepresent invention is used to modify glucosylated glycans in vitro. Inaddition, such soluble endomannosidase are purified according to methodswell-known in the art.

The secreted endomannosidases converts glucosylated structures (e.g.,GlcMan₅GlcNAc₂) FIG. 14( i) to deglucosylated structures (e.g.,Man₄GlcNAc₂) FIG. 14( ii) by hydrolyzing at least one glucose residueand one mannose residue on an oligosaccharide. For example, aglucose-α1,3-mannose dimer is cleaved from the glucosylatedoligosaccharide by the endomannosidase as shown in FIG. 14. Subsequentα1,2-mannosidase digestion FIG. 14( iii) results in the structure:Man₃GlcNAc₂ indicating an additional Manα1,2 on the trimannosyl core.

Host Cells

A number of host cells can be used to express the endomannosidase of thepresent invention. For example, the endomannosidase can be expressed inmammalian, plant, insect, fungal, yeast, algal or bacterial cells. Forthe modification of glucosylation on a protein of interest, preferredhost cells are lower eukaryotes producing Glc₁₋₃Man₉₋₅GlcNAc₂structures. Additionally, other host cells producing a mixture ofglucosylated glycans are selected. For example, a host cell (e.g., P.pastoris RDP25) producing the glucosylated structures such asGlcMan₅GlcNAc₂ in addition to unglucosylated structures such asMan₆GlcNAc₂ and its isomers is selected.

Preferably, a lower eukaryotic host cell is selected from the groupconsisting of Pichia pastoris, Pichia finlandica, Pichia trehalophila,Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum andNeurospora crassa.

Other hosts may include well-known eukaryotic and prokaryotic hosts,such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, andanimal cells, such as Chinese Hamster Ovary (CHO; e.g., thealpha-glucosidase I deficient strain Lec-23), R1.1, B-W and L-M cells,African Green Monkey kidney cells (e.g., COS 1, COS-7, BSC1, BSC40, andBMT10), insect cells (e.g., Sf9), and human cells (e.g., HepG2) andplant cells in culture.

Methods For Modifying Glucosylated N-Glycans

In another aspect of the invention, herein is provided a method formodifying the glucosylated glycans by introducing and expressing theendomannosidase of the present invention. FIG. 1, as highlighted, showsthe endomannosidase cleavage of the mono-, di-, and tri-glucosylatedglycans, represented by the second and third glucose residues.Accordingly, the endomannosidase enzyme of the present invention isintroduced into the Golgi of host (e.g. yeast) to enhance the efficiencyof deglucosylation, and thus enhancing subsequent trimming of the mannanstructure prior to the addition of further sugars to produce a morehuman-like N-linked glycosylation structure (FIG. 2).

In a further aspect of the invention, introduction of theendomannosidase into the Golgi (e.g. yeast) provides a method ofrecovering glucosylated glycoproteins that have entered the Golgi andare thus no longer accessible to the ER glucosidase I and II enzymes.The endomannosidase of the present invention can process suchglucosylated structures; for example, Glc₁₋₃Man₉₋₅GlcNAc₂ toMan₈₄GlcNAc₂, highlighted by the four mannose residues as shown in FIG.2. Accordingly, the present invention provides a quality controlmechanism wherein the recovered glucosylated oligosaccharides aredeglucosylated.

Moreover, it is contemplated that the use of the endomannosidaseobviates the need for the glucosidase I and II enzymes required in theearly steps of glycan trimming. In one embodiment, a host cell of thepresent invention may be deficient in glucosidase I and/or II activity.In the absence of glucosidase I or II activities, a host cell of thepresent invention may still exhibit a glucose catalyzing activitythrough the endomannosidase. Accordingly, herein is provided a method ofintroducing a nucleic acid encoding an endomannosidase into a host (e.g.yeast), upon expression, modifies glucosylated glycoproteins that haveentered the Golgi, which are a no longer accessible to the ERglucosidase I and glucosidase II enzymes. Preferably, the nucleic acidencoding the enzyme of the present invention cleaves a compositioncomprising at least one glucose residue and one mannose residue linkedto an oligosaccharide (FIG. 2). More preferably, a Glcα1,3Man dimer,Glc₂α1,3Man trimer or Glc₃α1,3Man tetramer are cleaved according to themethod of the present invention.

It will be a matter of routine experimentation for one skilled in theart to use the method described herein to optimize production ofdeglucosylated glycans (e.g. Man₄GlcNAc₂) using a selected fusionconstruct in a particular expression vector and host cell line.Accordingly, routine modifications can be made in the lower eukaryotichost cell expressing the endomannosidase of the present invention, whichconverts glucosylated glycans to deglucosylated glycans (e.g.Man₄GlcNAc₂) and subsequently to a desired intermediate for theproduction of therapeutic glycoproteins.

Introduction of Other Glycosylation Enzymes in Host Cells

Additionally, a set of modified glycosylation enzymes are introducedinto host cells to enhance cellular localization and activity inproducing glycoproteins of interest. This involves the fusion of varyinglengths of the catalytic domains to yeast endogenous targeting regionsas described in WO 02/00879. In one embodiment, a host cell P. pastorisYSH97 (och1 alg3 endmannosidase) is modified by introduction andexpression of glycosylation enzymes or catalytically active fragmentthereof selected from the group consisting of a 1,2-mannosidase I andII, GnT I (N-acetylglucosaminyltransferase I), GnT II, GnT III, GnT IV,GnT V, GnT VI, galactosyltransferase, sialyltransferase andfucosyltransferase. Similarly, the enzymes' respective transporters andtheir substrates (e.g. UDP-GlcNAc, UDP-Gal, CMP-NANA) are introduced andexpressed in the host cells. See WO 02/00879.

Endomannosidase pH Optimum

In another aspect of the invention, the encoded endomannosidase has a pHoptimum between about 5.0 and about 8.5, preferably between about 5.2and about 7.2 and more preferably about 6.2. In another embodiment, theencoded enzyme is targeted to the endoplasmic reticulum, the Golgiapparatus or the transport vesicles between ER, Golgi or the trans Golginetwork of the host organism, where it removes glucosylated structurespresent on oligosaccharides. FIG. 15 shows a pH optimum profile of thehuman endomannosidase (SEQ ID NO:2) (Example 9).

The following are examples which illustrate the compositions and methodsof this invention. These examples should not be construed as limiting:the examples are included for the purposes of illustration only.

EXAMPLE 1 Strains, Culture Conditions, and Reagents

Escherichia coli strains TOP10 or DH5a were used for recombinant DNAwork. Protein expression in yeast strains were carried out at roomtemperature in a 96-well plate format with buffered glycerol-complexmedium (BMGY) consisting of 1% yeast extract, 2% peptone, 100 mMpotassium phosphate buffer, pH 6.0, 1.34% yeast nitrogen base, 4×10⁻⁵%biotin, and 1% glycerol as a growth medium. The induction medium wasbuffered methanol-complex medium (BMMY) consisting of 1.5% methanolinstead of glycerol in BMGY. Minimal medium is 1.4% yeast nitrogen base,2% dextrose, 1.5% agar and 4×10⁻⁵% biotin and amino acids supplementedas appropriate. Restriction and modification enzymes were from NewEngland BioLabs (Beverly, Mass.). Oligonucleotides were obtained fromthe Dartmouth College Core facility (Hanover, N.H.) or Integrated DNATechnologies (Coralville, Iowa). MOPS, sodium cacodylate, manganesechloride were from Sigma (St. Louis, Mo.). Trifluoroacetic acid (TFA)was from Sigma/Aldrich, Saint Louis, Mo. The enzymes N-glycosidase F,mannosidases, and oligosaccharides were obtained from Glyko (San Rafael,Calif.). DEAE ToyoPearl resin was from TosoHaas. Metal chelating“HisBind” resin was from Novagen (Madison, Wis.). 96-welllysate-clearing plates were from Promega (Madison, Wis.).Protein-binding 96-well plates were from Millipore (Bedford, Mass.).Salts and buffering agents were from Sigma (St. Louis, Mo.). MALDImatrices were from Aldrich (Milwaukee, Wis.).

EXAMPLE 2 Cloning of Human and Mouse Endomannosidases

As a positive control, we amplified the region homologous to theputative catalytic domain of the rat mannosidase gene using specificprimers 5′-gaattcgccaccatggatttccaaaagagtgacagaatcaacag-3′ (SEQ ID NO:11) and 5′-gaattcccagaaacaggcagctggcgatc-3′ (SEQ ID NO: 12) andsubcloned the resultant region into a yeast integration plasmid usingstandard recombinant DNA techniques (See, e.g., Sambrook et al. (1989)Molecular Cloning, A Laboratory Manual (2^(nd) ed.), Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. and references cited therein, allincorporated reference; see also Example 3).

To identify the sequence of and isolate the ORF of the humanendomannosidase, we performed a protein BLAST search using the ratendomannosidase protein sequence (Genbank gi:2642187) and identified ahypothetical human protein (Genbank gi:20547442) of 290 amino acids inlength which shows 88% identity and 94% similarity to amino acids 162 to451 of the rat ORF (FIG. 3A). The DNA 5′-terminus of this human sequencewas analyzed using translated BLAST and another hypothetical humanprotein (Genbank gi: 18031878) was identified that possessed 95%identity over the first 22 amino acids of the search sequence but thenterminates in a stop codon (FIG. 3B). Reading-frame analysis of thissecond sequence indicated that 172 amino acids were in-frame upstream ofthe homologus region (FIG. 3C). Combining both these 5′ and 3′ regionsproduced a putative sequence with an ORF of 462 amino acids (FIG. 4) anda predicted molecular mass of 54 kDa.

To confirm that the two human sequences are one entire ORF, we designedprimers specific to the 5′-terminus of the gi:18031877 ORF and the3′-terminus of the gi: 205474410RF (5′-atggcaaagtttcggagaaggacttgc-3′(SEQ ID NO: 13) and 5′-ttaagaaacaggcagctggcgatctaatgc-3′ (SEQ ID NO: 14)respectively). These primers were used to amplify a 1389 bp fragmentfrom human liver cDNA (Clontech, Palo Alto, Calif.) using Pfu Turbo DNApolymerase (Stratagene, La Jolla, Calif.) as recommended by themanufacturers, under the cycling conditions: 95° C. for 1 min, 1 cycle:95° C. for 30 sec, 60° C. for 1 min, 72° C. for 2.5 min, 30 cycles; 72°C. for 5 min, 1 cycle. The DNA fragment produced was incubated with TaqDNA polymerase for 10 min at 68° C. and TOPO cloned into pCR2.1(Invitrogen, Carlsbad, Calif.). ABI DNA sequencing confirmed that bothof the human sequences identified by BLAST searching produced onecomplete ORF, this confirmed construct was named pSH131.

The endomannosidase gene from mouse may be similarly amplified andisolated. (See also, e.g., Sambrook et al. (1989) Molecular Cloning, ALaboratory Manual (2^(nd) ed.), Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., Innis et al. (1990) PCR Protocols: A Guide toMethods and Applications, Academic Press, New York, N.Y. and referencescited therein, all incorporated reference). The primers5′-atggcaaaatttcgaagaaggacctgcatc-3′ mEndo forward (SEQ ID NO: 15) and5′-ttatgaagcaggctgctgttgatccaatgc-3′ mEndo reverse (SEQ ID NO: 16) areused to generate the mouse full-length endomannosidase open readingframe.

EXAMPLE 3 Generation of Recombinant Endomannosidase Constructs andExpression

To generate a yeast secreted form of the human endomannosidase, a regionencoding the putative catalytic domain was expressed in the EasySelectPichia Expression kit (Invitrogen) as recommended by the manufacturer.Briefly, PCR was used to amplify the ORF fragment from 178 to 1386 basesfrom pSH131 using the primers hEndo Δ59 forward and hEndo Δstop reverse(5′-gaattcgccaccatggatttccaaaagagtgacagaatcaacag-3′ (SEQ ID NO: 11) and5′-gaattcccagaaacaggcagctggcgatc-3′ (SEQ ID NO: 12), respectively, withan EcoRI restriction site engineered into each). The conditions usedwith Pfu Turbo were: 95° C. for 1 min, 1 cycle; 95° C. for 30 sec, 55°C. for 30 sec, 72° C. for 3 min, 25 cycles; 72° C. for 3 min, 1 cycle.The product was incubated with Taq DNA polymerase, TOPO cloned and ABIsequenced as described above. The resulting clone was designated pSH178.From this construct, the human endomannosidase fragment was excised bydigestion with EcoRI and subcloned into pPicZαA (Invitrogen, Carlsbad,Calif.) digested with the same enzyme, producing pAW105. This constructwas transformed into the Pichia pastoris yeast strain GS115 suppliedwith the EasySelect Pichia Expression kit (Invitrogen, Carlsbad,Calif.), producing the strain YSH16. Subsequently, the strain was grownin BMGY to an OD₆₀₀ of 2 and induced in BMMY for 48 h at 30° C., asrecommended by the kit manufacturers.

To confirm that the isolated ORF was an endomannosidase, the previouslyreported rat liver endomannosidase was amplified and expressed inparallel as a positive control. Briefly, the fragment encoding aminoacids 49 to 451 of the rat endomannosidase, corresponding to theputative catalytic domain, was amplified from rat liver cDNA (Clontech)using the same conditions as described for the human endomannosidaseabove. The primers used were rEndo A48 forward and rEndo Astop reverse(5′-gaattcgccaccatggacttccaaaggagtgatcgaatcgacatgg-3′ (SEQ ID NO: 17)and 5′-gaattccctgaagcaggcagctgttgatcc-3′ (SEQ ID NO: 18), respectively,with an EcoRI restriction site engineered into each). The PCR productwas cloned into pCR2.1, sequenced and the resultant construct namedpSH179. Subsequently, the rat endomannosidase was subcloned into pPicZαA(Invitrogen, Carlsbad, Calif.) and expressed in GS115 (Invitrogen,Carlsbad, Calif.) as described above, producing pAW106 and YSH13.

To N-terminal tag recombinant human and rat endomannosidases, a doubleFLAG tag was engineered 3′ to the Kex2 cleavage site of the alpha matingfactor and 5′ to the EcoRI restriction used for endomannosidase cloningin pPicZαA, as follows. Briefly, the phosphorylated oligonucleotidesFLAG tag forward and FLAG tag reverse(5′-P-aatttatggactacaaggatgacgacgacaagg-3′ (SEQ ID NO: 19) and5′-P-aattccttgtcgtcgtcatccttgtagtccata-3′ (SEQ ID NO: 20)) were annealedas described in Sambrook et al. (1989), supra, and ligated into pPicZαAdigested with EcoRI and dephosphorylated with calf alkaline phosphatase.A construct containing two tandem FLAG tags in the correct orientationwas named pSH241. Subsequently, rat and human endomannosidases weredigested from pSH179 and pSH178 with EcoRI and ligated into pSH241,digested with the same enzyme. The resultant rat and humanendomannosidase constructs were named pSH245 and pSH246, respectively.Transformation of these constructs into GS115 (Invitrogen, Carlsbad,Calif.) produced the strains YSH89 and YSH90, respectively. Expressionof endomannosidase activities in these strains was studied as describedabove.

EXAMPLE 4 Expression of Rat Endomannosidases in P. pastoris

The catalytic domain of rat endomannosidase was amplified from pSH179using the primers rat Endomannosidase A48 AscI and rEndo PacI(5′-ggcgcgccgacttccaaaggagtgatcgaatcgacatgg-3′ (SEQ ID NO: 21) and5′-ccttaattaattatgaagcaggcagctgttgatccaatgc-3′ (SEQ ID NO: 22), encodingAscI and Pac restriction sites respectively). These primers were used toamplify a 1212 bp fragment from pSH 179 using Pfu Turbo DNA polymerase(Stratagene) as recommended by the manufacturers, under the cyclingconditions: 95° C. for 1 min, 1 cycle: 95° C. for 30 sec, 60° C. for 1min, 72° C. for 2.5 min, 30 cycles; 72° C. for 5 min, 1 cycle. The DNAfragment produced was incubated with Taq DNA polymerase for 10 min at68° C. and TOPO cloned into pCR2.1 (Invitrogen, Carlsbad, Calif.). ABIDNA sequencing confirmed that both of the human sequences identified byBLAST searching produced one complete ORF. This confirmed construct wasnamed pSH223. Subsequently, the rat endomannosidase fragment wasdigested from this construct and ligated into the yeast expressionvector pRCD259, giving the construct pSH229. The expression constructcontains the hygromycin selection marker; GAPDH promoter and CYC 1terminator, with the cloning sites NotI, AscI and PacI located betweenthese two regions; URA3 targeting integration region; and a fragment ofthe pUC19 plasmid to facilitate bacterial replication.

EXAMPLE 5 Expression Vectors and Integration

To express the rat endomannosidase proteins in yeast, the cDNA encodingthe catalytic domain was cloned into the expression vector pRCD259producing the vector pSH229 (See Example 4). Subsequently, cDNAsencoding Gls1(s), Van1(s) and Mnn11(m) leaders were cloned 5′ to thecDNA encoding the rat endomannosidase catalytic domain producing theplasmids pSH278 (rEndo A48 Gls1s leader), pSH279 (rEndo Δ48 Van1sleader) and pSH280 (rEndo A48 Mnn11m leader). Integration was confirmedby colony PCR with the resultant positive clones being analyzed todetermine the N-glycan structure of a secreted reporter protein.

EXAMPLE 6 Northern Blot Analysis

Tissue distribution of human endomannosidase transcript was determinedwith a human Multiple Tissue Northern blot (Clontech) representing 2 μgof purified poly A⁺ RNA from each of the tissues according to theinstructions of the manufacturer. The 547 bp human endomannosidase DNAprobe (843-1389) used was generated using the RadPrime DNA LabelingSystem (Invitrogen, Carlsbad, Calif.) and [³²P]dCTP. The results areshown in FIG. 8.

EXAMPLE 7 SDS-PAGE and Western Blotting

Media from the P. pastoris cultures were analyzed for endomannosidasesecretion by running samples on a 10% SDS-PAGE (Laemmli, U.K. (1970)Cleavage of structural proteins during the assembly of the head ofbacteriophage T4. Nature, 227, 680-685) using the Bio-Rad Mini-ProteanII apparatus. The proteins were then transferred onto a nitrocellulosemembrane (Schleicher & Schuell, Keene, N.H.). Recombinantendomannosidase was detected using the anti-FLAG M2 monoclonal antibodyin combination with a goat anti-mouse HRP-conjugated secondary antibodyand visualized with the ECL Western detection system (AmershamBiosciences) according to the manufacturer's instructions. Media fromGS115 (Invitrogen, Carlsbad, Calif.) was used as a control. The resultsare shown in FIG. 9.

EXAMPLE 8 In Vitro Characterization of Recombinant Endomannosidase

GlcMan₅GlcNAc₂, a substrate for endomannosidase assays, was isolatedfrom the och1 alg3 mutant strains RDP25 (WO 03/056914A1) (Davidson etal, 2003 in preparation). 2-aminobenzamide-labeled GlcMan₅GlcNAc₂ wasadded to 10 μl of culture supernatant and incubated at 37° C. for 8 h orovernight. 10 μl of water was then added and subsequently the glycanswere separated by size and charge using an Econosil NH₂ 4.6×250 mm, 5micron bead, amino-bound silica column (Altech, Avondale, Pa.) followingthe protocol of Choi et al, Proc. Natl. Acad. Sci. U.S.A.100(9):5022-5027 (2003).

EXAMPLE 9 pH and Temperature Optima Assays of Engineered Endoα-1,2-mannosidase

Fluorescence-labeled GlcMan₅GlcNAc₂ (0.5 μg) was added to 20 μL ofsupernatant adjusted to various pH (Table 2) and incubated for 8 hoursat room temperature. Following incubation the sample was analyzed byHPLC using an Econosil NH2 4.6×250 mm, 5 micron bead, amino-bound silicacolumn (Altech, Avondale, Pa.). The flow rate was 1.0 ml/min for 40 minand the column was maintained to 30° C. After eluting isocratically (68%A:32% B) for 3 min, a linear solvent gradient (68% A:32% B to 40% A:60%B) was employed over 27 min to elute the glycans (18). Solvent A(acetonitrile) and solvent B (ammonium formate, 50 mM, pH 4.5. Thecolumn was equilibrated with solvent (68% A:32% B) for 20 min betweenruns. The following table shows the amount (%) of Man₄GlcNAc₂ producedfrom GlcMan₅GlcNAc₂ at various pHs (FIG. 15, Table 2).

TABLE 2 pH Optimum of Human Endomannosidase pH % of Man4 4 0 4.5 0 5 4.55.5 29.6 6 51.4 6.5 52 7 41.3 7.5 30 8.5 20

The temperature optimum for human endomannosidase was similarly examinedby incubating the enzyme substrate with culture supernatant at differenttemperatures (room temperature, 30° C. and 37° C.), 37° C. being theoptimum.

EXAMPLE 10 Reporter Protein Expression, Purification and Release ofN-Linked Glycans Protein Purification

Kringle 3 (K3) domain, under the control of the alcohol oxidase 1 (AOX1)promoter, was used as a model protein. Kringle 3 was purified using a96-well format on a Beckman BioMek 2000 sample-handling robot(Beckman/Coulter Ranch Cucamonga, Calif.). Kringle 3 was purified fromexpression media using a C-terminal hexa-histidine tag (Choi et al.2003, supra). The robotic purification is an adaptation of the protocolprovided by Novagen for their HisBind resin. Briefly, a 150 uL (μL)settled volume of resin is poured into the wells of a 96-welllysate-binding plate, washed with 3 volumes of water and charged with 5volumes of 50 mM NiSO4 and washed with 3 volumes of binding buffer (5 mMimidazole, 0.5M NaCl, 20 mM Tris-HCL pH7.9). The protein expressionmedia is diluted 3:2, media/PBS (60 mM PO4, 16 mM KCl, 822 mM NaClpH7.4) and loaded onto the columns. After draining, the columns arewashed with 10 volumes of binding buffer and 6 volumes of wash buffer(30 mM imidazole, 0.5M NaCl, 20 mM Tris-HCl pH7.9) and the protein iseluted with 6 volumes of elution buffer (1M imidazole, 0.5M NaCl, 20 mMTris-HCl pH7.9). The eluted glycoproteins are evaporated to dryness bylyophilyzation.

Release of N-Linked Glycans

The glycans are released and separated from the glycoproteins by amodification of a previously reported method (Papac et al., Glycobiology8(5):445-54 (1998)). The wells of a 96-well MultiScreen IP (Immobilon-Pmembrane) plate (Millipore) were wetted with 100 uL of methanol, washedwith 3x150 uL of water and 50 uL of RCM buffer (8M urea, 360 mM Tris,3.2 mM EDTA pH8.6), drained with gentle vacuum after each addition. Thedried protein samples were dissolved in 30 uL of RCM buffer andtransferred to the wells containing 10 uL of RCM buffer. The wells weredrained and washed twice with RCM buffer. The proteins were reduced byaddition of 60 uL of 0.1M DTT in RCM buffer for 1 hr at 37° C. The wellswere washed three times with 300 uL of water and carboxymethylated byaddition of 60 uL of 0.1M iodoacetic acid for 30 min in the dark at roomtemperature. The wells were again washed three times with water and themembranes blocked by the addition of 100 uL of 1% PVP 360 in water for 1hr at room temperature. The wells were drained and washed three timeswith 300 uL of water and deglycosylated by the addition of 30 uL of 10mM NH₄HCO₃ pH 8.3 containing one milliunit of N-glycanase (Glyko). Afterincubting for 16 hours at 37° C., the solution containing the glycanswas removed by centrifugation and evaporated to dryness.

Miscellaneous: Proteins were separated by SDS/PAGE according to Laemmli(Laemmli 1970).

EXAMPLE 11 Matrix Assisted Laser Desorption Ionization Time of FlightMass Spectrometry

Molecular weights of the glycans were determined using a Voyager DE PROlinear MALDI-TOF (Applied Biosciences) mass spectrometer using delayedextraction. The dried glycans from each well were dissolved in 15 uL ofwater and 0.5 uL spotted on stainless steel sample plates and mixed with0.5 uL of S-DHB matrix (9 mg/mL of dihydroxybenzoic acid, 1 mg/mL of5-methoxysalicilic acid in 1:1 water/acetonitrile 0.1% TFA) and allowedto dry.

Ions were generated by irradiation with a pulsed nitrogen laser (337 nm)with a 4 ns pulse time. The instrument was operated in the delayedextraction mode with a 125 ns delay and an accelerating voltage of 20kV. The grid voltage was 93.00%, guide wire voltage was 0.10%, theinternal pressure was less than 5×10-7 torr, and the low mass gate was875Da. Spectra were generated from the sum of 100-200 laser pulses andacquired with a 2 GHz digitizer. Man₅GlcNAc₂ oligosaccharide was used asan external molecular weight standard. All spectra were generated withthe instrument in the positive ion mode. The estimated mass accuracy ofthe spectra was 0.5%.

EXAMPLE 12 A Combinatorial Library to Produce a Chimeric EndomannosidaseProtein

A library of human, mouse, rat and/or any combination of mixedendomannosidases characterized by catalytic domains having a range oftemperature and pH optima is generated following published procedures(see, e.g., WO 02/00879; Choi et al. 2003, supra and the publication ofU.S. application Ser. No. 10/371,877 (filed Feb. 20, 2003)). Thislibrary will be useful for selecting one or more sequences which encodea protein having endomannosidase activity that performs optimally inmodifying the glycosylation pattern of a reporter protein to produce adesired glycan structure when expressed in a lower eukaryotic host cellsuch as a yeast. It is expected to be advantageous to target thecatalytic domain of the endomannosidase to a specific cellularcompartment. The DNA combinatorial library approach (in-frame fusionbetween a targeting peptide and an enzymatic domain) enables one toidentify a chimeric molecule which expresses an endomannosidase activityin a desired or an efficient way in the host cell used for theselection. An endomannosidase sequence is expressed in a number ofexpression systems—including bacterial, yeast and mammalian cells, tocharacterize the encoded protein.

To generate a human-like glycoform in a host, e.g., a microorganism, thehost is engineered to express an endomannosidase enzyme (such as thehuman or mouse endomannosidase described herein) which hydrolyzes mono-,di- and tri-glucosylated high mannose glycoforms, removing the glucoseresidue(s) present and the juxta-positioned mannose (see FIG. 1). A DNAlibrary comprising sequences encoding cis and medial Golgi localizationsignals (and optionally comprising ER localization signals) is fusedin-frame to a library encoding one or more endomannosidase catalyticdomains. The host organism is a strain, e.g. a yeast, that is deficientin hypermannosylation (e.g. an och1 mutant) and preferably, providesN-glycans having the structure GlcNAcMan₅GlcNAc₂ in the Golgi and/or ER.(Endomannosidase can hydrolyze Glc₁₋₃Man₉₋₅GlcNAc₂ to Man₈₄GlcNAc₂, sothe preferred GlcNAcMan₅GlcNAc₂ structure is not essential). Aftertransformation, organisms having the desired glycosylation phenotype areselected. Preferably, the endomannosidase activity removes a compositioncomprising at least a glucose residue and one mannose residue on anoligosaccharide. An in vitro assay is used in one method. The desiredstructure is a substrate for the enzyme alpha 1,2-mannosidase (see FIG.2). Accordingly, single colonies may be assayed using this enzyme invitro

The foregoing in vitro assays are conveniently performed on individualcolonies using high-throughput screening equipment. Alternatively, alectin binding assay is used. In this case the reduced binding oflectins specific for terminal mannoses allows the selection oftransformants having the desired phenotype. For example, Galantusnivalis lectin binds specifically to terminal α-1,3-mannose, theconcentration of which is reduced in the presence of operativelyexpressed endomannosidase activity. In one suitable method, G. nivalislectin attached to a solid agarose support (available from SigmaChemical, St. Louis, Mo.) is used to deplete the transformed populationof cells having high levels of terminal α-1,3-mannose.

1-13. (canceled)
 14. A host cell comprising a nucleic acid encoding achimeric endomannosidase comprising the catalytic domain of anendomannosidase fused to a cellular targeting peptide to target theendomannosidase to the ER or Golgi of the host cell.
 15. The host cellof claim 14 wherein the host cell is a mammalian, plant, insect, fungal,yeast, algal or bacterial cell.
 16. The host cell of claim 14, whereinthe host cell is selected from the group consisting of Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum and Neurospora crassa.
 17. A method formodifying glycosylation structures in a lower eukaryote comprising:expressing an endomannosidase activity in the lower eukaryote a chimericendomannosidase comprising the catalytic domain of an endomannosidasefused to a cellular targeting peptide to target the endomannosidase tothe ER or Golgi of the lower eukaryote wherein the endomannosidaseactivity removes a composition comprising at least a glucose residue andone mannose residue on an oligosaccharide.
 18. The method of claim 17wherein the endomannosidase activity further comprises the activity oftruncating Glc₁₋₃Man₉₋₅GlcNAc₂ to Man₈₋₄GlcNAc₂ wherein Glcα1,3Man,Glc₂α1,3Man or Glc₃α1,3Man are removed.
 19. The method of claim 17wherein the endomannosidase activity comprises hydrolysis of acomposition comprising at least one glucose residue and one mannoseresidue on glucosylated glycans.
 20. The method of claim 17 wherein theendomannosidase introduced are targeted to the endoplasmic reticulum,the early, medial, late Golgi, trans Golgi network or any vesicularcompartment within the host organism.
 21. The method of claim 17 whereinthe endomannosidase is of host origin but has been modified by mutation,promoter strength or copy number to enhance activity.
 22. The method ofclaim 17 wherein the endomannosidase is secreted.
 23. The method ofclaim 17 wherein the host cell is a mammalian, plant, insect, fungal,yeast, algal or bacterial cell.
 24. The method of claim 17 wherein thelower eukaryote is selected from the group consisting of Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae,Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum and Neurospora crassa.
 25. A method formodifying glucosylated glycoproteins comprising introducing anendomannosidase activity into a lower eukaryotic host cell wherein uponexpression of the endomannosidase activity modifies a glucosylatedglycoprotein that has bypassed the ER.